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Best Grunge Bands
by Anthony Gramuglia 2 years ago in list / bands
Like all rock genres, the best grunge bands are full of variety that, while drawing from the same pool, take the genre in fascinating new directions.
Growing up in the 90s, Grunge Music was inescapable. The likes of Kurt Cobain elevated what was essentially an underground movement into a mainstay of the rock genre. Critics first praised the movement... then came to loathe it as a hundred garage bands emerged out of the woodwork to be the next Kurt Cobain... by literally doing nothing more than what he did.
Like all movements centered around a select few bands, it can be easy to dismiss a movement as derivatives of a few great bands. This is an incredibly dismissive attitude to have. Like all rock genres, grunge is full of variety that, while drawing from the same pool, takes the genre in fascinating new directions. Grunge's height may have been twenty-five years old (more depending on when you read this article), but that does not mean that the songs of our youths (or childhoods) are any less incredible than they were yesteryear – or, in some cases, a few songs you might never had heard of before... ?
Let's all just accept that no grunge Band list is complete without Nirvana. Hell, Kurt Cobain may be one of the most influential musicians of the 90s. Many people at the time might have thought that Nirvana was just a flash in the pan. A successful band in its day that would burn out fast.
Yet even years later, Nirvana remains hugely popular. Yes, the intense feeling in their songs did capture the zeitgeist of the early 90s teenage culture, but it's more than that. The music is harsh, rough, and unpolished – typical of grunge – while their lyrics are simply poetic. Harsh, rough, sometimes indecipherable poetry, but poetry all the same. If you haven't listened to Nirvana in awhile, or dismiss them as too mainstream, I urge you to revisit them. Really take in their music. It is worth your full attention.
Mentioning Kurt Cobain seems difficult without also mentioning Courtney Love. Courtney Love was Cobain's wife, and, sadly, often gets short-changed as just a part of Cobain's story. But the truth is that Courtney Love, even before marrying Kurt Cobain, had a significant musical career as the lead singer and guitarist for the band Hole.
Hole started in the late 80s as another early grunge band, and continued on through the 90s, following Love's marriage with Cobain. Hole's earlier work came across as very unrefined and rough – typical of grunge and punk at the time. As time went on, Hole moved away from the harsher punk aesthetic, and into softer, even pop sounds. Sadly, the band has been on and off again during the course of the 2000s and 2010s following the death of band members and personal disputes.
Oh, and their antics during their concerts came across as somewhat... bizarre. Specifically, Love's antics.
The only British band on this list, Bush is often described as a post-grunge band, inspired by the grunge movement following the initial wave. Many people have pointed out Bush's similarities to Nirvana – often shameless and unrepentant.
Though dismissed by critics at the time (and, to a lesser extent, even now), Bush proved to be an excellent band with tons of quality music. The problem was timing. Most grunge music that has had a lasting impact emerged in the late 80s or early 90s. Bush's first album hit stores in 94. While Sixteen Stone was successful, it marked the beginning of the end of the first wave of grunge music. Had they emerged just a little sooner, they might have struck a cord.
Sadly... no, no sadly. Bush still is doing pretty well. They just released an album in 2017.
Arguably the edgiest name for any grunge band, Alice in Chains has existed in some form since the 90s, though, following the 90s, the band has had numerous states of disarray due to publicized drug issues and the like.
Alice in Chains owes its success in part to the grunge music, though Jerry Cantrell, lead singer and songwriter of the band, claims the band draws more heavily from heavy metal. Indeed, as it moved away from the grunge scene, Alice in Chains took on a more metal feel. Because of this, the band is a great blend of metal and grunge aesthetics that really gives its songs a very unique feel.
Still, whether its grunge or metal, Alice in Chains remains a mainstay of the rocking scene.
Known in some circles as the Godfathers of Grunge, Screaming Trees predates the grunge movement, but rose to popularity atop its waves. Incorporating a punk aesthetic combined with some psychadelic elements, they managed to bend genres before Nirvana really pushed the grunge movement to the forefront of pop culture.
Screaming Trees saw its greatest success in the UK. However, toward the later half of the 90s, Screaming Trees ended up having trouble with their record label. This, along with other difficulties, lead to the band's collapse in the year 2000. But over the course of fifteen years, they released seven albums, and became one of the most successful underground rock bands of the 90s.
The Melvins
Another band that preceded the grunge movement, The Melvins proved a huge inspiration on bands like Nirvana and Soundgarden. From the early 80s to now, the band has entertained with their slower, more ambient tunes, spiced up by some idiosyncratic tendencies that really confused people at the time. "Are they a metal band like Black Sabbath? But they're too slow and stuff... so... what are they?"
Their sound became the cornerstone of what we now understand to be grunge. Of course, at the time, it was just The Melvin's unique style. Thankfully, the right young ones were listening, or else there would be nothing else like this great band's unique style.
Mudhoney is one of the longest enduring grunge bands around. Emerging from the remnants of the 80s rock band Green River, Mudhoney started up as Nirvana's contemporary, being one of the initial members of the Grunge music movement leading into the 90s.
To this day, they still are out performing. Their most recent album, Vanishing Point, was released as recently as 2013. So yes, they are a long lasting band with a long history and long catalogue of songs. For over 35 years of entertainment (counting their Green River days), they deserve credit.
During the early days of grunge, all the great bands came out of Seattle. This was where the underground music scene really had a chance to flourish, so a lot of great bands, drawing from one another, developed that unique grungy sound. Stone Temple Pilots came out of San Diego, pretty far from Seattle. It was the emergence of this band that said "Yeah, Grunge? Its scope of influence and impact is a lot larger than you thought before. It isn't just some Seattle thing."
Okay, admittedly, no one could possibly think that when "Smells like Teen Spirit" was being blasted from every radio, but the emergence of Stone Temple Pilots was an important step along the path for grunge's development.
Oh, and the band is really good, too.
Most people seem to think Pilots emerged after the success of Nirvana, but the band had existed under another name as early as 89. They insisted that they were not rip-offs, but rather drawing from the same influences of Nirvana. As time went on, they distanced themselves more and more from grunge... and then collapsed. They have been on-again, off-again since 2008.
The Smashing Pumpkins, whether the band likes it or not, is forever tied to the grunge movement. It owes it success in part to the grunge wave following Nirvana and Pearl Jam, though the band really owes more to gothic rock and psychedelic rock, with elements of electronica sprinkled in as the band returned following a break-up.
The Smashing Pumpkins had toured with Guns N' Roses and Red Hot Chili Peppers during the late 80s, opening for them, but only in the 90s did they become a main attraction – thanks to both their extensive touring and the grunge movement. They sounded just enough like grunge to be part of that movement, even though the band tried pretty hard to distance themselves from the movement.
Well, it hardly matters. Whether they like it or not, they are tied to grunge pretty inseparably. If you like grunge, you might want to look at the group that grew up alongside them.
Mother Love Bone has something of a tragic story. Lead singer Andrew Wood died of a drug overdose shortly before the release of their first studio album. It became their sole album. Form 1988 to 1990, they rocked Seattle... but then, nothing.
But that one album became a success following the band's collapse. The early 90s hunger for grunge led fans to uncover Mother Love Bone and their sole album, Apple. This tragic hidden gem never managed to find success in its own lifetime, but it has received a legacy worthy of its potential.
But, thankfully, that potential did not go untapped...
From the corpse of Mother Love Bone, a phoenix rose. While members of Mother Love Bone did collaborate with Soundgarden, it wasn't until Stone Gossard and Jeff Ament of Mother Love Bone joined Eddie Vedder and Mike McCready that Pearl Jam, one of the most commercially successful grunge bands... ever.
In many respects, Pearl Jam can be considered the band of the 90s. While the band undeniably started as typical, if not high-quality, grunge, as it went on it incorporated numerous other styles without ever losing its roots as a grunge band. In addition, Pearl Jam consistently went against the music industry, from refusing interviews to boycotting Ticketmaster. Behavior like this would have killed other bands hoping for mainstream success, but somehow, Pearl Jam survived.
The reason why? Because their music is awesome.
Another off-shoot of Mother Love Bone, this band lasted for only two years. Conceived as a tribute to the fallen lead singer of Mother Love Bone by Chris Cornell of Soundgarden, They only released one self-titled album that failed to meet success until a year after its release.
What makes Temple of the Dog such an oddity in the grunge scene is that it feels in many ways as a side-project for all parties involved. Everyone had moved on from Mother Love Bone, yet this one band – a tribute band – released some of the most underrated, quality music of the grunge movement. If you've overlooked this band, you owe it to yourself to give them a chance.
"Who is better? Nirvana or Soundgarden?"
Hard question, but I don't think it really matters what the answer is. Soundgarden, though less famous than Nirvana, is just as essential to the movement as Nirvana. It was the first grunge band to sign onto a major label, which already gives the band major bragging points.
But unlike Nirvana, with its unrefined, harsh sound, Soundgarden's music had structure. It came across as defined. While undeniably grunge, songs like "Black-Hole Sun" have a very distinct structure to it that makes it stand apart from its contemporaries.
Oh, and the music video for "Black-Hole Sun" may be among the weirdest music-videos to emerge out of the grunge music scene.
While the ban seemingly disbanded during the late 90s, it reunited in 2010 to go on tour.
listbands
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Anthony Gramuglia
Obsessive writer fueled by espresso and drive. Into speculative fiction, old books, and long walks. Follow me at twitter.com/AGramuglia
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Neoleptoneta bullis is een spinnensoort in de taxonomische indeling van de Leptonetidae.

Het dier behoort tot het geslacht Neoleptoneta. De wetenschappelijke naam van de soort werd voor het eerst geldig gepubliceerd in 2004 door Cokendolpher.

Leptonetidae

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Q: On bare-metal, non-OS, how context switching occurs upon exceptions/interrupts When CPU receives exception, Pre-processing by hardware
Saving the current PC and PSW values in RAM (or in control
registers in the case of the fast interrupt) and
Reading of the vector
Branching to the start of the exception handling routine is done.But, General purpose registers and control registers other than the PC and PSW that are to be used within the exception
handling routine must be preserved on the stack by user program code at the start of the exception handling routine. Reverse is repeated by user code and hardware upon returning from exception.
(Reference: Renesas Rx62n hardware manual, page 297, Chapter:Exceptions)
My question is where is this user code for context switching and how it is getting called?

A: As per the Renesas CC-RX compiler document, To declare any global function as ISR we need to specify it as
#pragma interrupt func
void func (void) { ... }
and compiler will generate code for it as:
func:
PUSHM R1-R3; Saves the registers used in the function.
....
(R1, R2, and R3 are used in the function)
....
POPM R1-R3; Restores the registers saved at the entry.
RTE
Additionally, we can also specify some other parameters such as:
fast interrupt (in this case PC and PSW will be saved in Backup BPC and BPSW by hardware, otherwise on stack)
acc save/restore
nesting enable
use limited registers in interrupt functions and don't generate instruction for saving/restoring R6-R13 registers.
This solves my doubt.

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text:
Metapedia est une encyclopédie en ligne
fondée sur le fonctionnement du logiciel MediaWiki et des interwikis. Située politiquement à l'extrême droite, Metapedia prône notamment, entre autres idéologies, le nationalisme blanc, la promotion de la race aryenne et le suprémacisme blanc.

Synopsis

Lancement
Le site se fait connaître en  pour être officiellement lancé le  via une première édition en langue suédoise. La version anglaise naît le  suivie, en , par les versions française et allemande.

Fonctionnement

Metapedia est un site collaboratif multilingue basé sur le logiciel MediaWiki. Se présentant comme une alternative à Wikipédia, il se présente — du moins, à l'origine — comme  vouée à la mouvance nationale, à ses idéaux, à son histoire et à ses membres, indépendamment des époques concernées ou de leurs répartitions géographiques. La ligne éditoriale du site se veut axée sur la  du public et ambitionne de participer à une  du savoir humain. Les idéologies en cours s'inscrivent dans la lignée de divers courants nationalistes et d'extrême droite qui, à leur tour, s'inspirent des théories propagées par Metapedia pour alimenter leurs propres discours.

Objectifs

Le site affirme que l'un de ses objectifs vise à « présenter une image plus équitable de la lutte pro-européenne ». Le contenu argue également s'adresser au plus grand nombre, soit autant au grand public qu'aux universitaires qui, jusqu'alors, selon Metapedia, seraient induits en erreur par l'induction constante d'informations tronquées et erronées, voire arbitrairement hostiles aux « vrais chercheurs » en quête de vérité. Parmi d'autres écueils, Metapedia récuse les positions contestataires exprimées par plusieurs instances nommément désignées parmi lesquelles figureraient le magazine britannique , lAnti-Defamation League, le Southern Poverty Law Center, le centre Simon-Wiesenthal ainsi que d'autres entités structurellement apparentées.

Logo

Le logo de Metapedia représente le visage d'un jeune Aryen, inspiré d'une sculpture de l'artiste allemand Arno Breker (1900-1991) intitulée .

Contenu

Le site compte plus de  répartis en seize langues : hongrois, allemand, espagnol, suédois, roumain, estonien, français, slovène, tchèque, portugais, norvégien, croate, danois, grec et néerlandais. Les sujets abordés, outre l'histoire de l'Europe et la mythologie nordique, s'attachent également à valoriser la musique nationaliste puisant ses origines dans le berceau de la « race aryenne ». Par ailleurs, le quotidien britannique The Register stipule qu'un bon pourcentage du contenu de Metapedia tend à vénérer Odin, entité chapeautant la doctrine de l'ordre blanc de Thulé. S'y ajoutent l'étude des runes et autres déclinaisons scolastiques dont les fondements préconisent une préférence élective en faveur de la race aryenne, selon les préceptes édictés par Heinrich Himmler. Le journal argentin  souligne également les éloges exprimés à l'adresse du führer Adolf Hitler auquel se joignent d'autres personnalités prépondérantes indissolublement liées au nazisme.

Classement

Le site Metapedia figure au  rang mondial de l'Alexa Internet (au mois de ).

Critiques

Selon le Bureau de la protection constitutionnelle de Rhénanie-du-Nord-Westphalie, les articles de Metapedia sont teintés de propos niant la Shoah et tendent conjointement à glorifier l'Allemagne nazie. C'est la raison pour laquelle le Département fédéral allemand des médias préjudiciables à la jeunesse qualifie la disponibilité de son accès public comme .

Néonazisme

Début 2007, six mois environ après le lancement de la version originale en suédois, Metapedia devient l'objet d'une attention soutenue de la part des médias nationaux en raison de sa ressemblance fonctionnelle avec le site Wikipédia. Pourtant, le contenu rédactionnel en diffère sensiblement. En effet, l'encyclopédie en ligne Metapedia ne tarit pas d'éloges sur l'Allemagne nazie et ses figures de proue ; elle catalogue ostensiblement les personnes d'appartenance juive dans les journaux ; elle répertorie la plupart des compagnies du pays en fonction de leur légation suédoise pure souche ou, inversement, relevant d'une affiliation juive apparentée.

Investigations

Les distinctions ostentatoires précitées conduisent le parquet suédois à ouvrir une enquête . Le chancelier de justice est amené à se prononcer sur l'existence de possibles infractions pénales relevant de l'incitation à la haine raciale et autres inculpations liées à d'éventuelles violations de la loi sur la protection des données. Après examen des particularités inhérentes au dossier, le magistrat décide  de mettre fin à son enquête, considérant qu'aucun élément à charge suffisamment probant ne peut être invoqué en l'état au regard du droit à la liberté de parole ou du respect relatif à la sphère privée.

Non-lieu

En , en réponse à une attention diligente accrue en direction du site Metapedia, le chancelier suédois de la justice en arrive à la conclusion que le contenu publiquement exposé affiche bel et bien une image positive d'Adolf Hitler. Il décide cependant de ne pas initier de nouvelles investigations à l'encontre de Metapedia, attendu que l'implication légale des activités erratiques pratiquées n'est pas clairement démontrée, puisqu'elle n'enfreint pas stricto sensu les articles juridiques du code pénal susceptibles de pouvoir être invoqués à dessein d'entamer des poursuites circonstanciées à l'encontre des dirigeants.

Technologie
La technologie utilisée par Metapedia recourt au logiciel libre MediaWiki qui est un moteur de wiki conçu en langage PHP autour d'une base de données MySQL. Le projet a été initié par  qui a présidé aux premières ébauches. Il travaille actuellement en collaboration avec Lennart Berg, sous la houlette de NFSE Media AB dont le siège se trouve à Linköping en Suède.

Hébergement

NFSE Media AB est responsable du contenu de l'ensemble des informations mises en ligne par Metapedia. L'adresse IP attribuée au site est reliée à la société américaine SoftLayer Technologies, Inc. basée à Dallas aux États-Unis. Le corollaire détenteur de la dénomination « Neuschwabenland Archivs » est également hébergé sur un serveur américain.

Bibliographie

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Notes et références

Notes

Références bibliographiques

Autres références

Traductions

Voir aussi

Articles connexes

MediaWiki
Encyclopédie en ligne
Antisémitisme
Racisme
Néonazisme
2006 en Suède
Négation de la Shoah
Site web multilingue
Loisir créatif
Culture libre
Contenu sous licence libre
Fondation en 2006
Suprémacisme blanc
Extrême droite sur Internet
Nationalisme blanc


For a better efficiency and inventory control, we created an app for the mobile grading of unloaded material. To run the app we recommend the outdoor smartphone CAT B15Q from Caterpillar that is connected with the RECY® database either by WLAN or 3G.
Require Recy 5 at least SP82, or Recy 6 at least 6.2.50. Update only in agreement with RECY SYSTEMS AG!


text:
Spontaneous cleavage of RNA in ternary complexes of Escherichia coli RNA polymerase and its significance for the mechanism of transcription
C K Surratt, S C Milan, and M J Chamberlin
PNAS September 15, 1991 88 (18) 7983-7987; https://doi.org/10.1073/pnas.88.18.7983
C K Surratt
S C Milan
M J Chamberlin
Ternary complexes of RNA polymerase, bearing the nascent RNA transcript, are intermediates in the synthesis of all RNAs and are regulatory targets of factors that control RNA chain elongation and termination. To study the catalytic and regulatory properties of RNA polymerases during elongation, we have developed methods for the preparation of these intermediates halted at defined positions along a DNA template. To our surprise, some of these halted complexes undergo a reaction in which the RNA transcript is cleaved up to 10 nucleotides from its 3'-terminal growing point. The 5'-terminal fragment, bearing a free 3'-OH residue, remains bound to the RNA polymerase-DNA complex and can resume elongation, whereas the 3'-terminal oligonucleotide of 2-10 nucleotides, bearing a 5'-phosphate, is released. RNA cleavage occurs only in the ternary complex and requires a divalent metal ion such as Mg2+. Since RNA polymerases are believed to have a single catalytic site for nucleotide addition, this reaction is unlikely to be due to hydrolysis catalyzed by this site comparable to the 3'----5' exonuclease activity associated with the catalytic center found for some DNA polymerases. Nor is this reaction easily explained by models for transcription elongation that postulate a 12-base-pair DNA.RNA hybrid as intermediate. Instead, we suggest that this is an unusual kind of protein-facilitated reaction in which tight binding of the RNA product to the enzyme strains the RNA phosphodiester linkage, resulting in cleavage of the RNA well away from the catalytic center. By this model, the nascent RNA enters a product binding site beginning 3 or 4 nucleotides from the growing point at the 3' terminus. This RNA binding site extends for up to 16 nucleotides along the protein surface. The stress brought about by this binding appears to vary considerably for different ternary complexes and may play a role in driving the translocation of the RNA polymerase along the DNA.
You are going to email the following Spontaneous cleavage of RNA in ternary complexes of Escherichia coli RNA polymerase and its significance for the mechanism of transcription
C K Surratt, S C Milan, M J Chamberlin
Proceedings of the National Academy of Sciences Sep 1991, 88 (18) 7983-7987; DOI: 10.1073/pnas.88.18.7983


Lexington, Ky. - Former Kentucky American Water Executive Nick Rowe has been tapped for a new leadership position within the utility's parent company. Rowe, who has been Senior Vice President of American Water's Eastern Division, today begins his new role leading the water company's Central Division based in St. Louis. Rowe will oversee the president's of Kentucky American Water and six other American Water operations in Illinois, Indiana, Iowa, Michigan, Missouri and Tennessee.
Mr. Rowe recently talked with 's Tom Martin about his tenure in Lexington and a number of issues related to politics, conservation and community.
TM: In a way this is an exit interview. You're going to be pulling up stakes and moving to St Louis.
NR: Yes, it is. But you know Lexington has been a big part of our lives for the last twelve years and me and my wife are both Bowling Green, Kentucky natives so it's kind of bittersweet. It's a great opportunity from a career standpoint but still a lot of good friends here and not only within the business but externally in our community as well.
TM: Is it difficult to pull up stakes and move to another community?
NR: Absolutely. When we moved here our kids were very small and as they get older they, you know we got them through high school here, grade school and middle school, my older daughter went to the University of Kentucky. So there are a lot of fond memories here and through that process of being involved in the community and civic organizations and the relationships that you've formed, it's just always a challenge for a family. But its part of corporate America today.
TM: What have you been doing with Kentucky American since Cheryl Norton stepped into the role of President? What's been your role?
NR: Well, you know my role as Senior Vice President for the division has been a supportive role of really trying to make sure we are doing the right things from an operating standpoint, from a financial standpoint and our corporate responsibilities standpoint, involved in our communities. But with Cheryl I've really taken a back seat and focused on the other states to allow her some room to grow and develop and she's done a great job of that and really getting out in the community and building her relationships and getting introduced to different folks and different organizations so we can continue to be a vital part of this community.
TM: So you are moving into the mid-section of the country, into the true Midwest, which of course will come with its own geographic issues and so forth that differ from those in the east. Can you kind of tell us about what you know about the difference?
NR: Well, I've traveled through that Midwest area quite a bit growing up as a kid from Bowling Green, Kentucky. The good news is they are similar to Lexington in how they treat people. Lexington is a great community as far as welcoming new people into the community and embracing them. The Midwest has a little bit of that same flavor, from what I can tell. Of course, St. Louis is a big metropolitan area, much larger than Lexington. There is some exciting things like pro teams and things of that nature and so we'll get a chance to take advantage of that. But overall I think the community is kind of what you make of it and everywhere we have lived whether it's been north, midwest or south we just make it our home and we intend to do the same thing there.
TM: Are water related issues different in any way?
NR: No, very similar. In Missouri and Illinois, big communities, high growth areas, water is a needed necessity for the economic prosperity. They haven't had some of the same challenges that we've had here in Lexington over the years. But overall water is pretty plentiful there with the rivers that they have.
TM: Okay, you mentioned challenges here in this market and one would relate to the recent mayoral election.
TM: In that election Kentucky American announced a significant 38 percent rate increase to support financing of a new plant on the Kentucky River. That was initiated prior to the election instead of awaiting PSC approval and it turns out that when PSC did act on the rate hike request, it reduced the increase to 29 percent. In his initial run for office Mayor Jim Newberry was a firm opponent of condemnation of the water company - which, of course, supported the position of Kentucky American, only to have this huge rate increase arise in the weeks prior to the election. Mr. Newberry has made no secret of his belief that this contributed to his failure to win reelection. Do you have any thoughts on that?
NR: Well, I don't know if that's true or not. You know, when you're in a business that has invested $164 million on a project that we're pretty proud of because it's been an issue that has plagued this community for decades. Over two decades we've been trying to get the water supply issue resolved and we got it done. You have to do what's right for the customers and I think we made the right decision. As far as the political side of that we really did not get into that discussion or thought process, we just knew we had to get the project done for the large industries that we have here, for the homeowners in giving them that flexibility to use water when they need to along with a pretty stringent conservation policy. So we felt we did the right thing. You know, it would be just like me or you or any customer building a brand new home and saying 'well, you know we're not going to pay the bank back for two or three years because this is a bad political time to do that.' We tried not to make that decision. We built the plant, we got it done and filed our rates based on what we thought was fair and reasonable. The commission ordered us to solve the problem and we got it done. And it's unfortunate the timing was around election time. When you can take that one issue and put it all in one basket, I doubt if that was the case. It's probably a lot of issues and some that probably will be debated for years to come.
TM: It might be viewed that the former mayor really stuck his neck out on behalf of Kentucky American in opposing condemnation, only to feel that he was undermined by that initial announcement of the 38 percent rate increase when PSC had not even approved it yet. I know there is a perception out there that it was a political decision, but you're saying it was not.
NR: No, no. We don't make decisions like that. I mean really our decision was solely based on the fact that we had made such a huge capital investment and it was time to start paying back the lenders for the loans and the debt service that we had taken on for that large project. Again to me it compares to a house note that payment comes due right away and we have to start paying for that and that's how we made our decision. And we have tried to stay out of that whole debate around the political issue. We were just focused on getting our project done and getting cost recovery.
TM: With the plant now up and running, what does that imply for the future for Lexington and its water supply?
NR: Well, it puts Lexington in a very comfortable position as we continue to grow our economy. Lexington has been very blessed that we have not taken as big a hit as other communities. If you think about communities just south of us like in Atlanta, which is a great community, a very large city that has outgrown its water needs. And they really struggled a couple of years ago. And most folks who are not in the industry are not aware of that. They've got a major water shortage issue that is being debated right now on how they are going to solve it because Atlanta has grown so quickly. The good news for Lexington is we've got the issue resolved for decades to come and as we attract new industry or expand plant facilities here, whatever the case may be, we're well positioned to move forward.
TM: How is this economy affecting your ability to make decisions about future growth?
NR: So far we haven't really seen a major shift in our customer demand. A little bit less industrial usage here and there and a little bit less manufacturing demand but nothing of a magnitude that would change our demand projections substantially.
TM: Is water a limited resource? Are we looking at a finite resource that we better be taking care of?
toilet facilities, the low flow faucets and things of that nature with the new plumbing codes. So we've seen people take on that conservation initiative. But you go out west and you ask for a glass of water, you've got to ask for it when you sit down at the table because they really conserve because so much of the western states depend on Hoover Dam and the Colorado River to supply that whole west coast area. So it is something that we can't be taking for granted and we should be stressing conservation for our kids and the next generation each and every opportunity. And we've done a lot of that I think in Lexington. I've seen folks really trying to do the right thing. But we've all got teenagers, we all have gone through the time when we have to ask them to cut back their showers and it lasts for a little while and we've got to keep reminding. It's just a different generation. My wife and I, you know we've got two daughters in the household where they take those showers and you've got to peck on the wall to get those ladies to shut it down after awhile. It's still hard to get that mindset when that 'why do we have to conserve the water' keeps coming out. It's going to take an educational process and we've tried to do that through a lot of our conservation programs here not only at Kentucky American but American Water in general. We've tried to do that throughout the nation.
TM: When do you make your move?
NR: Well, it's effective November 1 to start the new position. I'm excited by that but my family will be behind here. I've got a daughter who works at LexMark so she'll live here in this community. My wife is a nurse at Central Baptist Hospital so she'll stay here for quite a while and we may retain a home here for awhile and maybe get temporary living in St. Louis. We have a lot of good friends here. It's a community that we love and we'd love to retire here and we'll be back. And again the good news is that Kentucky is still part of my division so I'll be spending quite a bit of time with Kentucky employees. So, I'll still be about to see the employees, see friends, have a cup of coffee with them; play a round of golf with them when I can on the weekend or whatever the case may be. I'll be traveling quite a bit for the first six months. So my wife will probably transition there when she gets an opportunity.
TM: Nick Rowe, Executive with Kentucky American Water we appreciate you taking the time and the best of luck to you.
NR: Thank you Tom. Appreciate the interview.


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Obese mammary tumour-bearing mice are highly sensitive to doxorubicin-induced hepatotoxicity
Megan Sedeman1,2,
Claudia Christowitz1,2,
Louis de Jager3,4 &
Anna-Mart Engelbrecht1,2
BMC Cancer volume 22, Article number: 1240 (2022) Cite this article
Breast cancer is a major health burden for women, worldwide. Lifestyle-related risk factors, such as obesity and being overweight, have reached epidemic proportions and contributes to the development of breast cancer. Doxorubicin (DXR) is a chemotherapeutic drug commonly used to treat breast cancer, and although effective, may cause toxicity to other organs. The mechanisms and effects of DXR on hepatic tissue, and the contributing role of obesity, in breast cancer patients are poorly understood. The aim of this study was therefore to investigate the effects of DXR on hepatic tissue in an obese tumour-bearing mouse model.
A diet-induced obesity (DIO) mouse model was established, where seventy-four three-week-old female C57BL6 mice were divided into two main groups, namely the high fat diet (containing 60% kcal fat) and standard diet (containing 10% kcal fat) groups. After eight weeks on their respective diets, the DIO phenotype was established, and the mice were further divided into tumour and non-tumour groups. Mice were subcutaneously inoculated with E0771 triple negative breast cancer cells in the fourth mammary gland and received three doses of 4 mg/kg DXR (cumulative dosage of 12 mg/kg) or vehicle treatments via intraperitoneal injection. The expression levels of markers involved in apoptosis and alanine aminotransferase (ALT) were compared by means of western blotting. To assess the pathology and morphology of hepatic tissue, haematoxylin and eosin staining was performed. The presence of fibrosis and lipid accumulation in hepatic tissues were assessed with Masson's trichrome and Oil Red O staining, respectively.
Microscopic examination of liver tissues showed significant changes in the high fat diet tumour-bearing mice treated with DXR, consisting of macrovesicular steatosis, hepatocyte ballooning and lobular inflammation, compared to the standard diet tumour-bearing mice treated with DXR and the control group (standard diet mice). These changes are the hallmarks of non-alcoholic fatty liver disease, associated with obesity.
The histopathological findings indicated that DXR caused significant hepatic parenchymal injury in the obese tumour-bearing mice. Hepatotoxicity is aggravated in obesity as an underlying co-morbidity. It has been shown that obesity is associated with poor clinical outcomes in patients receiving neo-adjuvant chemotherapy treatment regimens.
Breast cancer is a common malignancy that frequently occurs in women from both developed and developing countries and is therefore a major health burden worldwide [1]. In 2020, breast cancer was the most frequently diagnosed cancer and the leading cause of cancer-related deaths among females worldwide [2]. Lifestyle-related risk factors, such as obesity and being overweight reached epidemic proportions and is a well-known risk factor that contributes to the development of breast cancer [3]. Dysfunctional adipocytes implicated in obesity is associated with altered secretion of metabolic substrates, adipokines, cytokines, growth factors, and inflammatory molecules which promote cell proliferation, metabolic reprogramming, angiogenesis, invasion and metastasis, and subsequently mediate tumour initiation, progression, and treatment response [4].
Significant progress has been made regarding treatment options for cancer patients, however, these drugs may cause serious multi-organ toxicity, and this remains a major concern. Doxorubicin (DXR) is an anthracycline glycoside antibiotic that possess antitumour activity and is one of the most effective chemotherapeutic agents used to treat breast cancer [5]. Although effective, therapeutic applications of DXR are limited due to its side-effects, such as myelosuppression, chronic cardiotoxicity, and skeletal muscle atrophy [6, 7]. DXR can also be detrimental to other organs, such as the liver, which plays an important physiological role in metabolism, glycogen and triglyceride storage, plasma protein synthesis and detoxification of toxic metabolites [8]. It has been shown that DXR can induce hepatotoxicity, which limits its efficacy in anticancer therapy, resulting in poor patient prognosis and decreased overall survival of cancer patients [9, 10].
DXR can accumulate in the reticuloendothelial system (RES) which is responsible for clearance of particles and substances in circulation and tissues [11]. The RES is a major site of liposome accumulation after systemic administration [11]. The liver is one of the primary organs associated with the RES and exhibits the largest capacity for liposome uptake. Therefore, a substantial amount of DXR can accumulate in liver tissue, making it more prone to DXR-induced toxicity.
DXR-induced oxidative stress is characterized by accumulation of reactive oxygen species (ROS), which decrease antioxidant defence systems, resulting in oxidative damage of deoxyribonucleic acid (DNA), therefore, mediating apoptotic cell death in hepatic tissue [10]. It has been shown that the apoptotic pathway, characterized by the release of cytochrome c from the mitochondria (intrinsic apoptotic pathway) and the Fas ligand (important regulator of the extrinsic apoptotic pathway), were associated with DXR-induced acute toxicity in the liver [10, 12]. Furthermore, histopathological changes have indicated that DXR can cause structural damage to hepatic tissues which include inflammation, congestion, and necrosis [10, 12, 13].
Non-alcoholic fatty liver disease (NAFLD), a co-morbidity associated with obesity is characterized by macrovesicular steatosis, triglyceride accumulation in hepatocytes, increased hepatic oxidative stress and increased sensitivity to drug induced liver injury [14]. A study done by Alghamdi et al. (2015) showed that DXR in the presence of palmitate and oleate (lipid loading) caused a significant accumulation of lipids within the human hepatoma cell line (Huh7 cells) and enhanced acute toxicity in lipid-loaded hepatocytes, which is mediated through increased oxidative stress and ROS [15].
The mechanisms of action on hepatotoxicity and the contributing role of obesity in breast cancer patients are not fully elucidated. Therefore, the aim of this study was to investigate the effects of DXR on hepatic tissue in an obese tumour-bearing model. The expression levels of markers involved in the apoptotic pathway was assessed by means of western blotting. Alanine transaminase (ALT) expression was also determined through western blotting. To assess the pathology and morphology of hepatic tissues, haematoxylin and eosin (H&E) staining was performed on formalin fixed paraffin embedded (FFPE) tissue. Furthermore, the presence of fibrosis and steatosis in hepatic tissues were assessed with Masson's trichrome and Oil Red O staining, respectively.
Ethical clearance was obtained from the Stellenbosch University animal research committee (no. SU-ACUM13-00,015 and no. ACU-2020–14,751). The experiments involving the use of laboratory animals were carried out in accordance with the Animal Welfare Act and recommendations of the Institutional Animal Care and Committee of Stellenbosch University. This study is reported in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) to improve the reporting of research involving animals.
Three-week-old female C57BL6 mice (n = 74) were obtained from the Stellenbosch University Central Research Facility and were housed at the Stellenbosch University Animal Unit in individually ventilated cages (IVC) at temperature-controlled conditions (i.e., 22ºC) and underwent a 12-h light/dark cycle. Mice were allowed an acclimatization period of one week with ad libitum access to mouse pellets and tap water (Fig. 1). The general welfare of the animals was monitored daily. After one week of acclimatization, seventy-four mice were randomly divided into two main groups, namely the high fat diet (HFD, n = 36) and standard diet (SD, n = 38) groups (Fig. 1). To induce obesity, mice were fed a HFD containing 60% kcal fat (D12492, OpenSource Diets®, Research Diets Inc., New Jersey, USA), whereas a SD containing 10% kcal fat (D12450J, OpenSource Diets®, Research Diets Inc., New Jersey, USA) were used to generate lean control mice (Supplementary data, Table S1) [16]. According to literature, C57BL/6 mice are sensitive to diet-induced obesity (DIO) [17]. Body weight was monitored weekly over the study period and mice were on their respective diets up until the end point of the study.
Summary of the in vivo model and respective experimental groups
After eight weeks on their respective diets, the DIO phenotype was established and the two groups were further randomly divided into two groups each, namely the tumour (T) and non-tumour (NT) groups. Mice were subcutaneously inoculated with E0771 triple negative breast cancer cells suspended in Hanks Balanced Salt Solution (HBSS) (Sigma Chemical Co., St Louis, MO, USA) in the fourth mammary pad, using a 23-guage needle syringe. The tumour groups were inoculated with E0771 triple negative breast cancer cells (HFD + T, n = 18; SD + T, n = 18) and the non-tumour groups were not inoculated with cancer cells (HFD + NT, n = 18; SD + NT, n = 20) (Fig. 1).
Once the tumours became palpable (200 – 300 mm2), DXR treatment was initiated. The mice were randomly divided into vehicle control (V, isovolumetric intra-peritoneal injection of HBSS) and DXR treatment (D5794, LKTR laboratories, Minnesota, USA) groups. Mice were restrained and three successive dosages of 4 mg/kg DXR were administered every three days (cumulative dosage of 12 mg/kg) via intraperitoneal injection. In humans, the dosage of 12 mg/kg DXR is equivalent to 36 mg/m2 and falls within the relevant dosage range of DXR treatment (15—90 mg/m2) administered to cancer patients in the clinical setting [18]. The eight experimental groups were assigned as follow: High fat diet + Tumour + Doxorubicin (HFD + T + DXR, n = 9); High fat diet + Non-Tumour + Doxorubicin (HFD + NT + DXR, n = 10); Standard diet + Tumour + Doxorubicin (SD + T + DXR, n = 10); Standard diet + Non-Tumour + Doxorubicin (SD + NT + DXR, n = 10), High fat diet + Tumour + Vehicle (HFD + T + V, n = 9); High fat diet + Non-Tumour + Vehicle (HFD + NT + V, n = 8); Standard diet + Tumour + Vehicle (SD + T + V, n = 8); Standard diet + Non-Tumour + Vehicle (SD + NT + V, n = 10) (Fig. 1).
The mice were weighed every second day and the last body weight was recorded on the day of euthanasia. Tumour growth was measured using a Harpenden caliper (mm) and individual tumour volumes were calculated according to the following equation [19]:
$$Tumour\;volume\;(mm^3)=\;\frac1{2\;\left(length\;x\;width^2\right)}$$
Fasting blood glucose, triglyceride and lactate levels were taken using the tail prick method where 5 μl of blood was collected on test strips using the Accu-Chek ® Performa Nano (Roche Diagnostics, Mannheim, Germany) and Accutrend Plus® (Roche Diagnostics, Mannheim, Germany) respectively. The mice were euthanized 3 days after the last DXR treatments were administered. The mice were anesthetized with 3% isoflurane (Isofor, Safeline, Pharmaceuticals, Florida, South Africa) and were euthanized by cervical dislocation. Hepatic tissues were excised, where half of the tissues were snap frozen in liquid nitrogen and stored at—80 °C for western blotting and Oil red O staining (n = 4). The other half of the tissues were preserved in 10% formalin for histological analysis (n = 4–5).
Blood plasma samples were used to quantify TNF-a, IL-6, IL-10, leptin (PPX-04-MXCE327, Thermo Fisher Scientific, United States) using a custom ProcartaPlex panel and matched mouse Luminex kit. Insulin was quantified using a Milliplex mouse adipokine magnetic bead panel MAP kit (MADKMAG-71 K, Burlington, Massachusetts, United States). All analyses were performed according to the manufacturers' protocols and specifications. Analytes were measured simultaneously using a MAGPIX system plate reader (APX1042, Bio-Rad, California, United States) and data (expressed in pg/ml) was processed on Bioplex Software 6.1 (Bio-Rad, California, United States).
Hepatic tissue samples were placed on ice and allowed to thaw. Samples were suspended in 300 μl cold modified radio-immunoprecipitation assay buffer (RIPA) containing protease and phosphatase inhibitors. Surgical scissors cleaned with 100% ethanol was used to cut tissues into smaller pieces while on ice. Samples were homogenised (KineMatica PolytronTM PT2100, Fisher Scientific) while on ice. Samples were centrifuged (14, 000 RCF (g), 20 min, 4 °C) to yield distinct layers and the supernatant layer was removed and transferred into sterile Eppendorf tubes. Samples were then centrifuged again at 14, 000 RCF (g), 20 min, 4 °C. The process of removing the supernatant was repeated followed by protein determination using a Bradford assay. Protein samples were prepared with Laemmli's sample buffer and were loaded onto 4–20% Criterion™ TGX Stain-Free™ Precast Gels (mini-PROTEAN® TGX™ Gels, Bio-Rad), following protein separation at 100 V for 10 min and 120 V for 60 min in Tris/Glycine/SDS running buffer (BioRad, CA, USA). Proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Trans-Blot Turbo RTA Midi PVDF transfer kit, BioRad, CA, USA) with the Trans-Blot Turbo Transfer System (BioRad, CA, USA) using mixed molecular weight. The membranes were blocked in 5% milk prepared in tris-buffered saline with tween 20 (TBS-T) for 2 h at room temperature (RT) and then incubated in primary antibody, at 4 °C overnight. On the following day, the membranes were incubated with secondary antibody for 1 h at RT. Primary and secondary antibody details are listed in the additional file (Supplementary data, Table S2). After incubation, the membranes were developed on the ChemiDoc™ MP System. Specific bands were visualized and detected using enhanced chemiluminescence (ECL) substrate detection (BioRad, CA, USA). Quantification of protein samples were normalized to total protein and expressed as a percentage of the control.
In all animals, a small portion of the right lateral lobe of the liver tissue was fixed in 10% neutral formalin buffered solution prior tissue processing (n = 4–5). Hepatic tissues were processed using an automated tissue processor (HistoCore PEARL, Leica Biosystems) on a 12-h cycle followed by infiltration with paraffin embedded wax (Leica EG 1150 H). Tissues were sectioned into 5 μm sections using a microtome (Leica RM 2125 RT) and tissue sections were placed onto positively charged histobond microscope slides. Two histochemical stains were carried out on the sectioned tissue; H&E stain for morphometric and pathological evaluation and Masson's trichrome stain to evaluate the presence of fibrosis within hepatic tissue. The right median lobe of hepatic tissue was snap frozen in liquid nitrogen and sectioned into 7 μm sections using a cryostat (Leica CM 3050 S Research Cryostat, Leica Biosystems) to evaluate the presence of lipid accumulation using Oil red O staining (n = 4–5). Sections were placed onto positively charged histobond microscope slides and left to defrost. Sections were stained in Oil red O in dextrin staining solution (Sigma-Aldrich, 01,391, SA) for 25 min. Coverslips were mounted onto the slides using aqueous mounting media (Sigma-Aldrich, G 0918) prior to imaging under a microscope (Nikon ECLIPSE E400). The relative number of red pixels (lipid droplets) were quantified using Image J software v1.52a.
Three individuals, blinded to the treatment allocations, scored five images per tissue sample in each treatment group using the non-alcoholic fatty liver disease (NAFLD) activity score (NAS). This is an accredited and validated scoring system frequently used to evaluate hepatic steatosis, inflammation, and hepatocyte-specific pathology [20]. Each scorer was provided with a grading sheet to familiarize themselves with the scoring system one day prior to analysis. The scores were tabulated and used for inter- and intra-observer analysis. H&E-stained tissues were used to evaluate structural changes that occurs within hepatocytes and Masson's trichrome-stained tissues were used to assist with the evaluation of liver fibrosis. Steatosis was scored according to its percentage per microscopic field, location (zone, 1, 2 or 3), and the presence of micro- and/or macrovesicular steatosis. With regards to inflammation, the location, lobular and/or portal, were evaluated per 100X microscopic field. Following H&E staining, microvesicular steatosis were indicated by black arrows and macrovesicular steatosis were indicated by red arrows (Fig. 5). Ten images per sample were taken and analysis was performed from right to left across the liver tissue sections. The H&E-stained and Masson's trichrome-stained liver tissues were evaluated as follow: To assess the extent of steatosis, the presence of hydropic changes, micro- and macrovesicular steatosis were evaluated. To determine whether inflammation occurred, the presence of portal mononuclear cell infiltration, haematopoiesis, and Kupffer cell proliferation were evaluated. Characteristics of hepatocytes and the portal system, such as hepatocyte swelling, sinusoidal and central vein dilation, were also evaluated.
The western blot experiments were conducted with biological repeats of n = 4 and technical repeats of n = 1. Bio-Rad Image Lab™ software v6.0.1 was used for normalization of the protein specific intensities against total protein intensities. For the Oil Red O staining, ten images per sample were quantified in Image J software v1.52a and the relative number of red pixels were analysed among the different treatment groups. Statistical analysis was performed using GraphPad Prism v7.0. To determine whether the data was normally distributed, a normality test was performed using the Shapiro-Wilks test. To describe the differences between two groups, a Mann Whitney t-test was used. A three-way ANOVA (analysis of variance) followed by Fishers LSD post hoc test was used to describe the differences between three/or more groups and to determine the relationship between the three variables present in this study, namely, diet, disease, and treatment. The results were reported as mean ± standard error of the mean (SEM) and p < 0.05 was considered statistically significant. Histological experiments were conducted with biological repeats of n = 4–5 and technical repeats of n = 2.
Establishing a diet-induced obesity phenotype
To establish whether obesity was induced, differences in body weights were determined between mice fed a SD and HFD for eight weeks, prior to tumour inoculation and DXR treatment (Fig. 2). After eight weeks on their respective diets, mice fed an HFD showed statistically significant higher body weights at week 6 (p < 0.01), week 7 (p < 0.001) and week 8 (p < 0.0001) compared to mice fed a SD (Fig. 2). We therefore conclude that the DIO phenotype was established after 8 weeks on their respective diets and continued throughout the study.
Body weight of female C57BL6 mice from week one to week eight on their respective diets. A Mann Whitney t-test was performed to compare the differences in body weight (grams) between the SD and HFD mice after eight weeks. Values were represented as mean ± SEM and p < 0.05 was considered statistically significant (SD, n = 38; HFD, n = 36). **—p < 0.01, ***—p < 0.001, ****—p < 0.0001. SD: standard diet; HFD: high fat diet
To further confirm the DIO phenotype, fasted blood parameters such as fasted glucose, triglyceride, lactate, and insulin levels were determined. Adipokines, such as leptin, IL-6 and TNF-α were also assessed. No statistically significant differences were observed for triglycerides (Fig. 3B), lactate (Fig. 3C), insulin (Fig. 3G), IL-6 (Fig. 3E), and TNF-α (Fig. 3F) levels between the different treatment groups. Fasting blood glucose (p = 0.043) (Fig. 3A) and leptin levels (p = 0.0267) (Fig. 3D) were significantly up regulated in the HFD group compared to the SD group.
Fasting blood parameters of mice fed a standard or high fat diet for ± 12 weeks. Mice were fasting for 12 h prior to euthanasia to determine (A) glucose, (B) triglycerides, (C) lactate, (D) leptin level (E) interleukin-6, (F) TNF-α and (G) insulin levels. Results are represented as mean ± SEM (n = 5–6). *—significantly different compared to the standard diet group (p < 0.05). **—significantly different compared to the standard diet group (p < 0.01)
Hepatic tissue weight and hepatic hypertrophy
Mice in the HFD + NT + V group showed a significantly higher hepatic tissue weight compared to the SD + NT + V mice (p = 0.0406) and SD + T + V mice (p = 0.0218) (Fig. 4). Mice in the HFD + NT + DXR group showed a significantly higher hepatic tissue weight compared to mice in the SD + T + V group (p = 0.0400) (Fig. 4).
Hepatic tissue weight (grams) between the different groups. Three-way ANOVA with Fishers LSD post hoc correction test was applied and p < 0.05 was considered statistically significant. Results are presented as mean ± SEM (n = 8–10 per group). *—significantly different compared to the SD + NT + V group (p < 0.05), #—significantly different compared to the SD + T + V group (p < 0.05). SD: Standard diet; HFD: High fat diet; T: Tumour; NT: Non-Tumour; DXR: Doxorubicin; V: Vehicle
Hepatotoxicity: Apoptotic cell death and ALT
For the purpose of this study, we focussed on the three objectives namely, 1) the effects of obesity on hepatotoxicity, 2) the effects of DXR treatment on hepatotoxicity, and 3) the effects of obesity on the outcomes of DXR-induced hepatotoxicity. All western blot results are included in the supplementary data set.
Apoptotic cell death
To determine whether apoptosis was induced in the hepatic tissue samples, western blot experiments were performed to compare the protein expression levels of different apoptotic markers between the different treatment groups. For the intrinsic apoptotic pathway, no significant differences were observed in caspase-9 protein expression between the different treatment groups (supplementary data, Fig. S1). We also assessed the effector caspase, where no significant differences were observed in caspase-3 protein expression between the different treatment groups (supplementary data, Fig. S4). Furthermore, no significant differences were observed in cleaved PARP protein expression between the different treatment groups (supplementary data, Fig. S5). There were also no significant differences observed in ALT expression between the different treatment groups (supplementary data, Fig. S6).
Histopathology of hepatic tissue (H&E staining)
Using the accredited NAFLD activity score (Table 1), macrovesicular steatosis was observed in the HFD + NT + DXR group with > 33%-66% steatosis present (grade 2). The HFD + T + DXR group showed extensive hepatocyte ballooning and sinusoidal dilation with > 66% steatosis present (grade 3) compared to the SD + NT + V (control group) where areas of steatosis alternated with areas of normal morphology (Table 1). Macrovesicular steatosis were therefore observed in the HFD + T + DXR group (indicated by red arrow) accompanied with mild inflammation (indicated by white arrow) and sinusoidal dilation (indicated by yellow arrow) and congestion (Fig. 5). Vacuoles of lipids are accumulating in the hepatocytes in the HFD + T + DXR group and are displacing the nucleus to the cell's periphery (indicated by red arrow) (Fig. 5). Inflammatory foci (mild inflammation) were observed in the SD + T + V, HFD + NT + V and HFD + T + DXR groups (indicated by white arrow) with < 2 foci per 200 × field (grade 1) (Fig. 5, Table 1).
Table 1 Liver pathology evaluated by the nonalcoholic fatty liver disease activity score [20, 21]
Examples of pathological lesions observed in H&E-stained hepatic tissue in mice fed a HFD evaluated by the non-alcoholic fatty liver disease activity scoring system (NAS) (n = 4–5). Microvesicular steatosis (indicated by black arrows) and macrovesicular fatty infiltration in hepatocyte (steatosis) (indicated by red arrows), mild inflammation (inflammatory foci) (indicated by white arrows) and sinusoidal dilation (indicated by yellow arrow) within hepatocytes between the different groups. Images were taken at 20 × magnification, scale bar = 100 μm
Macrovesicular steatosis was minor in the HFD + NT + V group (indicated by red arrow) and accompanied with mild inflammation (indicated by white arrow) (Fig. 5). Macrovesicular steatosis was moderate in the HFD + NT + DXR group (indicating in red arrow), which indicates that DXR aggravates the toxicity in hepatic tissue (Fig. 5). Macrovesicular steatosis was minor in the HFD + T + V group (indicated by red arrow) (Fig. 5). Microvesicular steatosis with < 5% steatosis within hepatocytes occurred in the SD + NT + V control group (grade 0), accompanied with no inflammatory foci (grade 0) and none ballooning present (grade 0) (indicated by black arrow) (Fig. 5, Table 1). Microvesicular steatosis with > 33%-66% steatosis within hepatocytes were present in the SD + T + DXR group (grade 2), accompanied with no inflammatory foci (grade 0) and few balloon cells (grade 1) (indicated by black arrow) (Fig. 5, Table 1). Macrovesicular steatosis with 5%-33% steatosis within hepatocytes (grade 1), accompanied with inflammatory foci (grade 1) (indicated by white arrow) and non-ballooning were observed in the HFD + NT + V group, where areas of steatosis > 33%-66% were observed in the HFD + NT + DXR group with no inflammatory foci present (grade 0) (Fig. 5, Table1). Macrovesicular steatosis 5%-33% were observed within hepatocytes in the HFD + T + V group (grade 1) (indicated by red arrow) (Fig. 5, Table 1). To summarise; our results demonstrated that macrovesicular steatosis was observed in mice fed a HFD and microvesicular steatosis was observed in mice fed a SD.
Oil red O staining
To evaluate the presence of lipid accumulation within hepatocytes, Oil Red O staining was performed (Fig. 7). To detect the amount of lipid droplets among the different treatment groups, the relative number of red pixels were analysed using Image J (Fig. 6). This method was used by Masone et al., 2017 [22] & Gojanovich et al., 2016 [23] to accurately quantify and analyse size distribution of cellular lipid droplets. The HFD + T + V group showed a highly statistically significant increase in the relative number of red pixels (lipid droplets) compared to the SD + T + V group (p = 0.0057). The HFD + T + DXR group indicates a highly statistically significant increase in the relative number of red pixels (lipid droplets) compared to the SD + T + DXR (p = 0.0023) and the HFD + NT + V control group (p = 0.0318). Hepatocytes stain amphophilic and lipid droplets are stained red (indicated by black arrows) (Fig. 7). High levels of lipid accumulation were observed within hepatocytes in the HFD groups. Furthermore, lipid accumulation was significantly increased in the HFD group treated with DXR and the tumour present, which indicates that lipid accumulation was more severe in the HFD + T + DXR group compared to the SD + T + DXR group (Figs. 6 and 7).
Relative number of red pixels indicating the amount of lipid droplets within hepatocytes (n = 4–5). Results are presented as mean ± SEM. Three-way ANOVA with Fishers LSD post hoc correction test was applied and p < 0.05 was considered statistically significant. *—Significantly different compared to the Standard diet + Tumour + Doxorubicin (SD + T + DXR) group (p < 0.05), **—Significantly different compared to the SD + T + DXR group (p < 0.01), ##—Significantly different compared to the Standard diet + Tumour + Vehicle (SD + T + V) group (p < 0.01). SD: Standard diet; HFD: High fat diet; T: Tumour; NT: Non-Tumour; DXR: Doxorubicin; V: Vehicle
Representative images of lipid accumulation in hepatic tissue detected by the Oil Red O stain (n = 4–5). Lipid droplets (indicated by black arrows). Images were taken at 20 × and 10 × magnification, scale bar = 100 μm
Masson's trichrome stain
To evaluate liver damage, collagen fibres are stained blue using Masson's trichrome stain to determine the presence of fibrosis. No significant fibrosis was observed within the treatment groups, and therefore, no trends or statistical significance could be determined (Fig. 8). Images were qualitatively represented.
Representative images of liver tissue stained by the Masson's trichrome stain for pathological evaluation of fibrosis. (n = 4–5). Images were taken at 20 × and 40 × magnification, scale bar = 100 μm
Establishment of an obese model following a high fat diet
In animal models, a HFD is associated with an increase in body mass (BM) [24,25,26,27] and liver mass, which was observed in the HFD groups within this study. To establish whether obesity was induced, differences in body weight was evaluated for mice fed a SD or HFD for eight weeks prior to tumour inoculation and DXR treatment (Fig. 2). The body weights of mice in the HFD group were significantly higher compared to the SD group (Fig. 2), which confirmed that the obese phenotype was successfully established in our in vivo model. Our results correlate with similar findings of a study done by Lee et al. (2019) [28] and Santander et al. (2015) [29] who indicated that a HFD increased body weight, as a result of increased fat mass in C57BL6 mice by evaluating body composition. It has been shown that the C57BL6 mice strain is highly susceptible to the development of DIO and therefore correlates with the findings of our study [17].
To further establish the DIO phenotype, fasted blood parameters such as fasted blood glucose (Fig. 3A), triglycerides (Fig. 3B), lactate (Fig. 3C), and insulin levels (Fig. 3G) were determined. Adipokines, such as leptin (Fig. 3D), IL-6 (Fig. 3E) and TNF-α (Fig. 3F) were also assessed. While no statistically significant differences were observed for triglycerides, lactate, IL-6 and TNF-α levels, fasting blood glucose (p = 0.043) and leptin (p = 0.0267) levels were significantly up-regulated in the HFD group compared to the SD group. No significant differences were observed in the insulin levels between the different groups (Fig. 3G). Obesity is associated with insulin resistance which can promote the development of type 2 diabetes mellitus (T2DM). Therefore, the elevated fasting blood glucose levels in the HFD group is possibly indicative of insulin resistance associated with T2DM, clinically evidenced as a hyperglycemic state. The increased leptin levels observed in the HFD group, further confirmed the state of obesity. Leptin, secreted by adipocytes, increases with higher degrees of adiposity which prevents the uptake of glucose, inhibits lipolysis, and impairs lipogenesis. In hepatocytes, leptin induces insulin-like effects by regulating the insulin signalling pathway. Furthermore, elevated levels of leptin are overexpressed at a gene level in adipose tissue and are associated with leptin resistance, which is implicated in obesity, inflammation, and breast tumourigenesis [3]. Therefore, the increased leptin levels observed in the high fat diet group, further confirm the state of obesity.
Furthermore, the DIO phenotype observed in mice fed a HFD was corroborated by a significantly higher hepatic tissue weights observed in the HFD + NT + V and HFD + NT + DXR groups compared to the SD + NT + V and SD + T + DXR groups, respectively (Fig. 4). Our results correlate with a study conducted by Jung et al., 2013 [30] where C57BL6 mice fed a HFD for nine-weeks resulting in an increase in BM and liver mass gain.
HFD and DXR promote macrovesicular steatosis in hepatocytes, but not fibrosis
As previously mentioned, the first structural changes that occurs when the liver is damaged is hydropic changes, where fluid filled vacuoles accumulates within the hepatocytes. It has been shown that a HFD can induce lipid accumulation via a process known as hepatic steatosis [27, 31]. Microvesicular steatosis was observed in the SD groups since hepatocytes are filled with tiny lipid droplets and the nucleus is located centrally in the cell. Macrovesicular steatosis was observed in the HFD groups where large fat droplets occupy the cytoplasm of hepatocytes, pushing the nucleus to the periphery (Fig. 5).
From the non-alcoholic fatty liver disease activity score (Table 1), > 66% steatosis was present within hepatocytes in the HFD + T + DXR group accompanied with lobular inflammation (score 2) and prominent ballooning (score 2), sinusoidal dilation and congestion, fatty infiltration, and ballooning of hepatocytes compared to the SD + NT + V (control) group where areas of steatosis alternated with areas of normal morphology (Fig. 5). In the SD + T + DXR group, micro-vesicular steatosis was observed, with 5%-33% steatosis in the hepatocytes. In the HFD + NT + DXR group, > 33%-66% of the hepatocytes had steatosis compared to the HFD + NT + V group. This indicates that DXR promotes hepatic steatosis in obesity.
Our histopathology observations correlate with previous studies conducted where a HFD and DXR treatment induced macrovesicular steatosis, ballooned hepatocytes, inflammatory cell infiltration and sinusoidal dilation, respectively [12, 27, 29, 32]. No significant fibrosis was observed in our experimental groups (Fig. 8). This indicates that the liver did not progress to cirrhosis, indicative of end-stage liver disease and that the highly metabolic organ had the capacity to regenerate itself or reverse the harmful stimulus in the broad spectrum of liver disease. This could also be attributed to the concentration and frequency of the DXR treatment as observed by other studies [32].
HFD promotes lipid accumulation within hepatocytes and induce hepatotoxicity in combination with DXR treatment
The liver plays an important role in lipid metabolism and stores fatty acids in the form of triglycerides. It has been shown that a HFD promotes lipid accumulation within hepatocytes via hepatic steatosis resulting in disturbances in lipid metabolism [27, 31]. Due to image evaluation of Oil Red O-stained fresh tissue sections, our results indicated that the relative number of red pixels are highly significantly increased in the HFD groups compared to mice fed a SD (Fig. 6). In the present study, the relative number of red pixels are highly significantly increased in the HFD + T + DXR compared to the SD + T + DXR group (Fig. 6). Our results correlate with previous studies conducted by Layman et al., 2019 [27] & Tsuru et al., 2020 [33] where a HFD induced hepatic lipid accumulation and inflammation within hepatocytes of mice fed a HFD, resulting in hepatic steatosis. It is evident from our H&E and Oil Red O results that lipid accumulation was elevated in the HFD groups and specifically severe in the HFD + T + DXR group, which indicates that obesity and the tumour present in combination with DXR treatment induced hepatic steatosis, which is a hallmark of NAFLD (Figs. 5 and 7). We therefore conclude that hepatic steatosis (> 66% steatosis) occurred in the obese tumour-bearing mouse model, resulting in NAFLD. Our findings correlate with a study done by Layman et al., 2019 [27] where mice fed a HFD induced hepatic steatosis, within hepatocytes.
Our histopathology results indicated that hepatic tissues of mice fed a high fat diet and treated with DXR displayed severe histological damage, such as macrovesicular steatosis, sinusoidal dilation, and lobular inflammation. This indicates that more hepatic toxicity occurred in mice fed a high fat diet and treated with DXR. We therefore conclude that hepatic steatosis, a hallmark of NAFLD occurred in the obese tumour-bearing mouse model. There was no evidence of fibrosis which indicates that the livers did not progress to cirrhosis. This study showed that hepatotoxicity is aggravated in obesity as an underlying co-morbidity in breast cancer patients receiving adjuvant chemotherapy, such as DXR (Fig. 9). It has been shown that obesity is associated with poor clinical outcomes in patients receiving neo-adjuvant chemotherapy treatment regimens [34]. Lower doses of DXR are prescribed for obese cancer patients to reduce side effects and adverse toxicities, which could compromise drug efficacy and contribute to the development of resistance [34,35,36]. This study, therefore, provided clear evidence of the effects of DXR and the contributing role of obesity in the context of breast cancer.
Summary of schematic representation of DXR-induced hepatotoxicity in an obese-tumour bearing mouse model. Demonstrating the effects of diet-induced obesity, breast tumours and DXR treatment on the outcomes of hepatotoxicity. Diet-induced obesity resulted in an increase in adiposity, body weight, and hepatic tissue weight in mice fed a HFD. Diet-induced obesity in the presence of breast tumours treated with DXR caused significant structural modifications to hepatic tissue and exacerbated the hepatoxicity in the obese tumour bearing mice. There was no evidence of fibrosis which indicates that the livers did not progress to cirrhosis
The datasets generated and analysed during the current study are not publicly available due datasets being too large to include but are available from the corresponding author on reasonable request.
ALT:
Alanine transaminase
Animal Research: Reporting of In Vivo Experiments
Aspartate aminotransferase
BM:
Body Mass
DNA:
DIO:
Diet-induced obesity
DXR:
ECL:
Electrochemiluminescence
FFPE:
Formalin fixed paraffin embedded
HBSS:
Hank's balanced salt solution
H&E:
Haematoxylin & eosin
HFD:
High fat diet
IVC:
Individually ventilated cages
IL-6:
Interleukin-6
JNK:
Jun N-terminal kinase
MCP-1:
Monocyte chemoattractant protein-1
NAFLD:
NAS:
Non-alcoholic fatty liver disease activity score
NF-kB:
Nuclear factor kappa-light-chain-enhancer of activated B cells
NT:
Non-Tumour
PVDF:
Polyvinylidene difluoride
RES:
Reticuloendothelial system
RIP1:
Receptor-interacting protein 1
RIPA:
Radio-immunoprecipitation assay buffer
Standard diet
SDS:
TBS-T:
Tris-buffered saline tween
T2DM:
Topoisomerase II
Vechile
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The authors wish to acknowledge Bianca Bock and Zaakiyah Emjedi for performing the animal model and Mr. Reggie Williams for assistance with the histology staining and Dr. Chantelle Venter for guidance with the histology Oil red O quantification.
This study was financially supported by research grants from the Cancer Association of South Africa (CANSA); South African Medical Research Council (SAMRC); and the National Research Foundation (NRF).
Department of Physiological Sciences, Stellenbosch University, Stellenbosch Campus, Stellenbosch, South Africa
Megan Sedeman, Claudia Christowitz & Anna-Mart Engelbrecht
Department of Global Health, Faculty of Medicine and Health Sciences, African Cancer Institute (ACI), Stellenbosch University, Cape Town, 8000, South Africa
Division of Anatomical Pathology, Stellenbosch University and National Health Laboratory Service (NHLS), Tygerberg Hospital, Cape Town, 8000, South Africa
Louis de Jager
Anatomical Pathology, PathCare, Cape Town, South Africa
Megan Sedeman
Claudia Christowitz
Anna-Mart Engelbrecht
All authors have read and approved the manuscript. MS performed the western blot techniques, analysed, and interpreted the results as well as the histological results. MS was also a major contributor in writing the manuscript. CC co-supervised this study and reviewed the final manuscript. LDJ reviewed the final manuscript. AME supervised this study and reviewed the final manuscript.
Correspondence to Megan Sedeman.
Ethical clearance for the in vivo study was obtained from the Stellenbosch University Ethical Committee (no. SU-ACUM13-00015 and no. ACU-2020–14751). Institutional and international ethical guidelines were applied with respect to the handling of experimental animals. All methods were performed in accordance with the relevant guidelines and regulations of the Animals (Scientific Procedures) Act 1986. This study is reported in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments) to improve the reporting of research involving animals.
The authors declare they have no competing interest.
Additional file 1: Fig. S1.
Original blots of total protein and caspase-9 (n = 1-4). Fig. S2. Original blots of total protein and caspase-8 (n = 1-4). Fig. S3. Original blots of total protein and cleaved caspase-8 (n = 1-4). Fig. S4. Original blots of total protein and caspase-3 (n = 1-4). Fig. S5. Original blots of total protein and cleaved PARP (n = 1-4). Fig. S6. Original blots of total protein and ALT (n = 1-4). Table S1. Dietary composition of the standard and high fat diets [16]. Table S2. Primary and secondary antibodies details.
Sedeman, M., Christowitz, C., de Jager, L. et al. Obese mammary tumour-bearing mice are highly sensitive to doxorubicin-induced hepatotoxicity. BMC Cancer 22, 1240 (2022). https://doi.org/10.1186/s12885-022-10189-z
DOI: https://doi.org/10.1186/s12885-022-10189-z
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