Protective Effects of Omega-3 Supplementation against Doxorubicin-Induced Deleterious Effects on the Liver and Kidneys of Rats

Anthracycline doxorubicin (DOX) is still widely used as a chemotherapeutic drug for some solid tumors. Although DOX is highly effective, its side effects are limiting factors, such as cardio, nephro and hepatotoxicity. As such, approaches used to mitigate these adverse effects are highly encouraged. Omega 3 (ω-3), which is a class of long-chain polyunsaturated fatty acids, has been shown to have anti-inflammatory and antioxidant effects in preclinical bioassays. Thus, we evaluated the protective effects of ω-3 supplementation on hepatotoxicity and nephrotoxicity induced by multiple DOX administrations in rodents. Male Wistar rats (10 rats/group) were treated daily with ω-3 (400 mg/kg/day) by gavage for six weeks. Two weeks after the first ω-3 administration, the rats received DOX (3.5 mg/kg, intraperitoneal, 1×/week) for four weeks. DOX treatment reduced body weight gain increased systemic genotoxicity and caused liver-related (increase in serum ALT levels, thickness of the Glisson’s capsule, compensatory proliferation and p65 levels) and kidney-related (increase in serum urea and creatinine levels, and incidence of tubular dilatation) deleterious outcomes. In contrast, ω-3 supplementation was safe and abrogated the DOX-related enhancement of systemic genotoxicity, serum urea and creatinine levels. Furthermore, ω-3 intervention reduced by 50% the incidence of kidney histological lesions while reducing by 40–50% the p65 protein level, and the proliferative response in the liver induced by DOX. Our findings indicate that ω-3 intervention attenuated the DOX-induced deleterious effects in the liver and kidney. Therefore, our findings may inspire future mechanistical investigations and clinical interventions with ω-3 on the reported outcomes.


Introduction
According to the World Health Organization (WHO), in 2019, cancer was ranked as the leading cause of death before 70 in several countries, with an estimated 19.3 million new cases and 10 million cancer deaths worldwide in 2020 [1,2]. In contrast, the development of different oncological therapies, including chemotherapy, is generally linked to a better quality of life and increased survival [3]. Chemotherapy is considered an advance in cancer treatment, but side effects are still a limiting barrier to the efficacy of this clinical intervention [3]. Doxorubicin (DOX), an anthracycline produced by Streptomyces peucetius bacterium, has been widely used since the 1970s as a chemotherapeutic drug for solid tumors (ovary, breast and gastrointestinal) due to its high efficacy [4,5]. The antitumor effect

The ω-3 Supplementation Attenuates a DOX-Induced Increase in Kidney Damage Markers
The DOX regimen adopted led to a significant decrease in final body weight, body weight gain, liver weights (absolute and relative), and relative weight of left and right kidneys (p < 0.001, for all) (Table 1) compared to control counterparts, as a consequence of known systemic DOX-induced toxicity [13,22]. Although ω-3 attenuated the DOXinduced decrease in absolute liver weight (p < 0.001), this supplementation did not modify the DOX-related effects in general (Table 1). Regarding serum markers, DOX increased ALT (p = 0.003), urea and creatinine (p < 0.001, for both) levels. Both markers of kidney function/damage were reduced by ω-3 supplementation (p < 0.001) ( Figure 1A). Data presented as mean ± standard deviation. N = the number of rats/group. Wistar rats received ω-3 (400 mg/kg body weight/day) by intragastrical administrations for six weeks (or vehicle). After two weeks of ω-3 treatment, animals received weekly single intraperitoneal injections of DOX (3.5 mg/kg body weight) for four weeks (or vehicle). The letters correspond to the statistical difference among groups by one-way ANOVA followed by a posteriori Tukey test (p < 0.05).
Molecules 2023, 27, x FOR PEER REVIEW 3 of 15 ALT (p = 0.003), urea and creatinine (p < 0.001, for both) levels. Both markers of kidney function/damage were reduced by ω-3 supplementation (p < 0.001) ( Figure 1A). Representative photomicrographs of nucleoids are also presented. Data are presented as mean ± standard deviation. N = 5 (serum) or 8 (genotoxicity) rats/group. Male Wistar rats received ω-3 (400 mg/kg body weight/day) by intragastrical administrations for six weeks (or vehicle). After two weeks of ω-3 supplementation, animals received weekly single intraperitoneal injections of DOX (3.5 mg/kg body weight) for four weeks (or vehicle). The letters correspond to the statistical difference among groups by one-way ANOVA followed by a posteriori Tukey test (p < 0.05). Vectors (yellow) interconnecting the medians depict the statistical modulation of DOX-induced effects by ω-3.  Representative photomicrographs of nucleoids are also presented. Data are presented as mean ± standard deviation. N = 5 (serum) or 8 (genotoxicity) rats/group. Male Wistar rats received ω-3 (400 mg/kg body weight/day) by intragastrical administrations for six weeks (or vehicle). After two weeks of ω-3 supplementation, animals received weekly single intraperitoneal injections of DOX (3.5 mg/kg body weight) for four weeks (or vehicle). The letters correspond to the statistical difference among groups by one-way ANOVA followed by a posteriori Tukey test (p < 0.05). Vectors (yellow) interconnecting the medians depict the statistical modulation of DOX-induced effects by ω-3.

The ω-3 Supplementation Attenuates a DOX-Induced Increase in Blood Genotoxicity
The DOX regimen adopted also led to a marked increase in peripheral blood genotoxicity compared to the control and/or ω-3 counterparts, as demonstrated by Tail Intensity (p = 0.022) and Tail Moment parameters (p = 0.03) ( Figure 1B). Of note, ω-3 supplementation significantly reduced the DOX-induced increase in both parameters, indicating a reduction in DNA damage in peripheral blood cells ( Figure 1B). However, the ω-3 supplementation alone did not alter any of the above-cited parameters (Table 1, Figure 1A,B), eliciting the safety of this short-term intervention.

The ω-3 Supplementation Reduced DOX-Induced Kidney Tubule Dilatation
In HE-stained sections, histopathological analysis of the kidneys revealed focal areas of tubular dilatation in both cortexes (including the Bowman's space) and medulla due to the appearance of hyaline (or eosinophilic proteinaceous) casts in the lumen of tubules-indicating an increased glomerular permeability often associated with glomerular damage [23]-as depicted in representative photomicrographs ( Figure 2). The DOX regimen increased the incidence of these alterations, regardless of their manifestation (cortex and/or medulla), compared to the control group (p < 0.001). The ω-3 supplementation significantly reduced, by 50% (80% to 30%), the occurrence of this histological alteration in DOX-submitted rats (p < 0.001) ( Figure 2).
No marked histopathological alterations were identified in liver parenchyma in response to DOX and/or ω-3 interventions after a histopathological analysis of the HEstained sections (Supplementary Data S1). Nonetheless, a thickening of hepatic Glisson's fibrous capsule was observed in Sirius-red stained sections in both DOX-treated groups (p < 0.001) ( Figure 3) in response to DOX-induced peritonitis, which is a known side effect of this drug in rats [24]. Representative photomicrographs of HE-sections showing capsule thickening are also shown in Supplementary Data S1. DOX also led to a slight but significant increase in collagen in the portal areas (p < 0.001), although no marked fibrous expansions were observed. DOX also tended to increase collagen in the stroma of the kidney cortex and medulla (Supplementary Data S2). The ω-3 supplementation did not alter these DOX-induced effects in the liver and kidneys (Figure 3, Supplementary Data S2). The animals supplemented with ω-3 alone for 6 weeks showed no histopathological alterations, reinforcing the safety of this intervention on these organs.  . DOX-treated rats showed focal areas of tubular dilatation in both cortexes (black arrows), including Bowman's space (red arrow) and medulla (black arrows), due to the appearance of hyaline (or eosinophilic proteinaceous) casts in the lumen of tubules. (E) Effects of omega 3 (ω-3) supplementation on doxorubicin (DOX)-induced effects on the incidence of kidney tubular dilatation. N = 10 rats/group. Male Wistar rats received ω-3 (400 mg/kg body weight/day) by intragastrical administrations for six weeks (or vehicle). After two weeks of ω-3 supplementation, animals received weekly single intraperitoneal injections of DOX (3.5 mg/kg body weight) for four weeks (or vehicle). * Statistical difference compared to the control group by Fisher's Exact test (p < 0.05). ** Statistical difference compared to DOX group by Fisher's Exact test (p < 0.05).
No marked histopathological alterations were identified in liver parenchyma in response to DOX and/or ω-3 interventions after a histopathological analysis of the HEstained sections (Supplementary Data S1). Nonetheless, a thickening of hepatic Glisson's fibrous capsule was observed in Sirius-red stained sections in both DOX-treated groups (p < 0.001) ( Figure 3) in response to DOX-induced peritonitis, which is a known side effect of this drug in rats [24]. Representative photomicrographs of HE-sections showing capsule thickening are also shown in Supplementary Data S1. DOX also led to a slight but significant increase in collagen in the portal areas (p < 0.001), although no marked fibrous expansions were observed. DOX also tended to increase collagen in the stroma of the kidney cortex and medulla (Supplementary Data S2). The ω-3 supplementation did not alter these DOX-induced effects in the liver and kidneys ( animals supplemented with ω-3 alone for 6 weeks showed no histopathological alterations, reinforcing the safety of this intervention on these organs. les 2023, 27, x FOR PEER REVIEW intragastrical administrations for six weeks (or vehicle). After two weeks of ω-3 supplement animals received weekly single intraperitoneal injections of DOX (3.5 mg/kg body weight) fo weeks (or vehicle). * Statistical difference compared to the control group by Fisher's Exact te 0.05). ** Statistical difference compared to DOX group by Fisher's Exact test (p < 0.05).

The ω-3 Supplementation Reduced a DOX-Induced Increase in Hepatocyte Proliferation
The DOX regimen adopted increased hepatocyte proliferation (p = 0.0004 demonstrated by Ki-67 immunostaining ( Figure 4). The ω-3 supplementation was ab revert this DOX-mediated indirect effect on the liver (Figure 4), which may be trigg as a compensatory response to DOX-induced hepatocyte damage, as indicate increased ALT serum levels. Both DOX and ω-3 did not affect cell proliferation of tu epithelial cells in the kidney cortex and medulla (Supplementary Data S3).

The ω-3 Supplementation Reduced a DOX-Induced Increase in Hepatocyte Proliferation
The DOX regimen adopted increased hepatocyte proliferation (p = 0.0004), as demonstrated by Ki-67 immunostaining ( Figure 4). The ω-3 supplementation was able to revert this DOX-mediated indirect effect on the liver (Figure 4), which may be triggered as a compensatory response to DOX-induced hepatocyte damage, as indicated by increased ALT serum levels. Both DOX and ω-3 did not affect cell proliferation of tubular epithelial cells in the kidney cortex and medulla (Supplementary Data S3).

The ω-3 Supplementation Reduced a DOX-Induced Increase in Hepatic p65 Protein
The DOX protocol adopted led to a 4-fold increase in the protein levels of the hepatic p65 subunit with a pro-inflammatory transcription factor NFκB-which is also closely related to a damage/inflammation-induced hepatocyte survival/proliferative response [25]and this effect was attenuated by ω-3 intervention (p = 0.029) ( Figure 5). However, the ω-3 supplementation could not modify the DOX-induced increase in hepatic Nrf2 protein levels (p = 0.0063), which may have been elevated in response to increased drug-induced oxidative stress. Although both p65 and Nrf2 had increased slightly in the kidney by DOX, the groups were not statistically different ( Figure 5).

The ω-3 Supplementation Reduced a DOX-Induced Increase in Hepatic p65 Protein
The DOX protocol adopted led to a 4-fold increase in the protein levels of the hepatic p65 subunit with a pro-inflammatory transcription factor NFκB-which is also closely related to a damage/inflammation-induced hepatocyte survival/proliferative response [25]-and this effect was attenuated by ω-3 intervention (p = 0.029) ( Figure 5). However, the ω-3 supplementation could not modify the DOX-induced increase in hepatic Nrf2 protein levels (p = 0.0063), which may have been elevated in response to increased druginduced oxidative stress. Although both p65 and Nrf2 had increased slightly in the kidney by DOX, the groups were not statistically different ( Figure 5).

Discussion
Our preclinical bioassay evaluated the effects of ω-3 supplementation on the DOXinduced toxic impact on the kidney and liver of male Wistar rats. In contrast to other recent rodent bioassays based on a single DOX administration [26][27][28], our design was based on multiple i.p. doses, resembling first-line DOX-based human chemotherapy regimens [29,30]. In brief, DOX increased systemic genotoxicity and caused liver-related (increase in serum ALT levels, Glisson's capsule thickness, compensatory proliferation and p65 levels) and kidney-related (increased serum urea and creatinine levels, and incidence of tubular dilatation) deleterious outcomes. Considering that irreversible cardiotoxicity and gonadotoxicity are addressed as the main side effects of DOX therapy [5,31,32], liver and kidney outcomes are usually neglected or poorly investigated/reported in clinical practice. Since these are essential organs for DOX metabolism and clearance, our findings reinforce the importance of screening DOX-induced hepatotoxicity and nephrotoxicity.
In contrast to these effects, ω-3 supplementation was safe and abrogated the DOXrelated enhancement of genotoxicity, urea and creatinine. Furthermore, ω-3 intervention reduced the incidence of tubular dilatation in the kidney while it reduced p65 protein levels and the toxicity-induced proliferative response in the liver. Preclinical studies investigating possible approaches towards mitigating cardiotoxicity have been significantly encouraged recently [33][34][35][36]. Our preclinical findings on ω-3 may inspire future clinical studies on attenuating kidney and liver outcomes.
Regarding liver-related outcomes, after four weeks of DOX administrations, DOXtreated rats developed a thickened fibrous capsule and increased collagen in the portal areas, similar to previous findings in mice [37]. Glisson's capsule thickening is attributed to DOX-induced peritonitis [24], while increased collagen in vascular structures may be associated with the pharmacokinetics of this anthracycline. DOX is predominantly metabolized by the hepatocytes, after entering (by diffusion) the portal system, to the major metabolite, the quinone doxorubicinol (DOXOL), and several cytotoxic aglycone metabolites. However, most of the DOX administrated (~50%) is excreted from the body into the bile without being biotransformed [38][39][40]. In the hepatocytes, DOXOL generation, which is catalyzed by the enzyme nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) reductases, leads to the production of reactive oxygen species (ROS), such as superoxide radicals and secondary reactive species, as lipid hydroperoxides [40]. These compounds cause direct hepatic damage and death, culminating in the leakage of hepatic enzymes, such as ALT [41]. Hepatocyte death triggers an increase in p65 levels and compensatory proliferation, as indicated by higher Ki-67 indices. In addition, DOX-mediated oxidative stress activates Nrf2-mediated antioxidant system response, as shown by increased protein levels of this transcription factor. In general, this DOX-mediated background impairs the normal energetic metabolism of hepatic tissue [41], which could lead to decreased body weight gain.
In contrast to these hepatotoxic effects, ω-3 reduced the hepatic p65 levels and hepatocyte proliferation, although this supplement did not modify hepatic Nrf2 levels. According to the Human Equivalent Dose (HED), using an allometric dose translation [42], our 400 mg/kg bwt ω-3 intervention is equivalent to the human dose of 65 mg/kg bwt, or ã 4 g daily dose for 60 kg humans, that equals 4 capsules of the commercial formulation used in our study. As briefly mentioned, p65 pathway activation is related to a pro-inflammatory response and stimulates a hepatocyte survival/proliferative response under damage or inflammation [25]. The ω-3 supplementation seems to negatively modulate downstream inflammatory responses mediated by NFκB in different tissues under injury stimuli, including the liver [43,44]. Previous findings indicate that ω-3 alleviates p65-induced inflammation through a Sirt1-mediated mechanism in hepatocytes. The nuclear translocation of nuclear p65 was significantly decreased after ω-3 treatment, while this effect was reversed after treatment with EX-527, a Sirt1 inhibitor [43]. p65 attenuation may reduce its downstream responses, including the proliferative stimuli. Considering that the development of therapyrelated malignant neoplasms involving a sustained cell proliferation hallmark is of the most deleterious DOX side effects [5], this ω-3 effect may also be beneficial in this aspect, although further investigation is required.
DOX and its reactive metabolites reach the systemic circulation after the first pass in the liver. Then it reaches the other organs. Therefore, monitoring DOX and DOX-OL levels in circulation is proposed to indicate DOX toxicity for cancer patients [45]. We found that DOX enhanced peripheral blood DNA damage, indicating increased systemic genotoxicity in response to DOX-induced oxidative stress. On the other hand, ω-3 supplementation decreased this DOX-mediated effect. In a similar bioassay, a preventive ω-3 supplementation (400 mg/kg bwt/day) for 30 days decreased the serum oxidative stress (malondialdehyde) induced by a single DOX administration while increasing serum reduced glutathione levels [21]. As such, in addition to DOX and DOX-OL determination in circulation, genotoxicity assessment in DOX bioassays should also be encouraged, enabling the early screening of potential preventive treatments.
Regarding kidney-related effects, DOX led to increased serum creatinine and urea levels and an augmented incidence of tubular dilatation due to eosinophilic proteinaceous casts. Nonetheless, DOX did not alter renal collagen deposition, epithelial cell proliferation and Nrf2/p65 levels in this organ. Although most DOX is excreted via bile, some DOX and its metabolites reach the kidneys and are excreted through the urinary system [46]. The serum and histopathological alterations indicate increased glomerular permeability often associated with glomerular damage [23]. Indeed, other studies suggest that DOX-induced oxidative stress and free radical formation of ferric anthracycline can be highly responsible for this direct nephrotoxic effect [47]. The supplementation of ω-3 reduced creatinine, urea levels and tubular dilatation occurrence. This effect does not seem to be associated with Nrf2 and p65 transcription factors, as they were unmodified by ω-3 intervention, although glomerular permeability-related mechanisms still need investigating.
The main limitation of our study, along with other recent bioassays, is that an experimental model has not been included in which DOX was used to treat animals with preexisting cancer [34]. Studies on the combined antitumoral effects of omega-3 and DOX are scarce, but ω-3 PUFA was shown to enhance DOX antitumoral effects in vivo (a Mat-BIII mammary adenocarcinoma tumor-bearing rat model) and in vitro (human A549 lung carcinoma and MCF-7 breast adenocarcinoma cell lines), partly by enhancing DOX concentration [48,49]. Moreover, as DOX is known to induce bone marrow suppression, the roles of this hematotoxic outcome on DOX-induced liver and kidney effects should be further explored, as the administration of bone marrow-derived mesenchymal stem cells alleviated DOX-induced liver injury in rats by improving the oxidative status and limiting apoptotic cell death [50]. Our findings indicate that our preclinical translatable ω-3 intervention attenuated DOX-induced deleterious effects on the liver and kidney. In addition to these beneficial effects, ω-3 supplementation is considered safe since this intervention had no marked biochemical, morphological, or molecular outcomes in both organs in the absence of DOX treatment. Therefore, our findings may inspire future mechanistical investigations and clinical interventions.

Experimental Design
Seven-week-old male Wistar rats (n = 40 animals) were randomly allocated into four groups (n = 10 rats/group, Figure 6). Initially, the ω-3 and DOX + ω-3 groups were treated with ω-3 [400 mg/kg of bw/day, Essential Nutrition, São José, Brazil] [20,21] by intragastrical (ig) administration for six weeks, while other groups received tap water (Figure 1). Doses of ω-3 were based on a previous rat bioassay showing cardioprotective effects against DOX-induced cardiotoxicity [20]. controlled temperature (22 ± 2 °C) and light/dark cycle 12:12 h. Body weight, water and food consumption, and the condition of the animals' health were all monitored and recorded twice a week. This animal study was performed under the approval of the Botucatu Medical School (FMB/UNESP) Ethics Committee on Use of Animals (CEUA) (1295/2019). All animals received humane care according to the criteria described in the "Guide for the Care and Use of Laboratory Animals" [52].

Genotoxicity Assessment
Four hours after the last injection of DOX, peripheral blood samples were collected from the periorbital plexus for the genotoxicity assessment using alkaline single cell gel electrophoresis (comet) assay, as previously established [53]. Blood samples were quickly mixed with low melting point agarose (100 µL 0.75% in PBS, Invitrogen, Waltham, MA, USA.) and spread onto slides pre-coated with normal point agarose (1.5% in PBS, Invitrogen, Waltham, MA, USA) and covered with coverslips. Following agarose solidification (4 °C for 5 min), coverslips were carefully removed. The slides were incubated with cold lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, 1% N-lauroyl-sarcosine, 1% Triton X-100 and 10% DMSO, pH 10) overnight, at 4 °C. Subsequently. The slides were washed three times in PBS and immersed in a fresh cold alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na2EDTA, pH > 13) for 20 min. Electrophoresis was conducted for 20 min at 1 V/cm (300 mA) for 20 min. The slides were then neutralized with 0.4 M Tris (pH 7.5), dehydrated in 100% ethanol, and stained with SYBR Gold solution (1:10,000) (Invitrogen, USA). Fifty randomly selected nucleoids were After two weeks of ω-3 supplementation, animals from DOX and DOX + ω-3 groups received weekly single intraperitoneal (ip) injections of DOX (3.5 mg/kg bw) for four weeks (cumulative dose of 14 mg/kg bw) ( Figure 6). Doses of DOX were based on Xue et al. [49] and Spivak et al. [51] regimens, given that the cumulative dose is the maximally tolerated dose for rats. Therefore, ω-3 was administered before (2 weeks) and during (4 weeks) the DOX regimen. After 6 weeks, animals were euthanized per decapitation under anesthesia (sodium thiopental, 120 mg/kg, ip). Blood samples were collected and centrifuged (1500× g, 10 min), and the serum was stored at −20 • C for biochemical assays. During necropsy, the liver and kidneys were removed, washed in saline solution 0.9% and weighed. Liver samples (lateral left lobe) and slices from both kidneys were collected and fixed in buffered formalin 10% or stored at −80 • C. After fixation, samples from both organs were kept in 70% alcohol until histological processing. Animals received drinking water and chow (Nuvital, Nuvilab, Brazil) ad libitum. In addition, animals were kept in propylene cages with stainless steel covers and pine wood shavings for bedding in a room that had continuous ventilation (16-18 air changes/hour), relative humidity (55 ± 10%), controlled temperature (22 ± 2 • C) and light/dark cycle 12:12 h. Body weight, water and food consumption, and the condition of the animals' health were all monitored and recorded twice a week. This animal study was performed under the approval of the Botucatu Medical School (FMB/UNESP) Ethics Committee on Use of Animals (CEUA) (1295/2019). All animals received humane care according to the criteria described in the "Guide for the Care and Use of Laboratory Animals" [52].

Genotoxicity Assessment
Four hours after the last injection of DOX, peripheral blood samples were collected from the periorbital plexus for the genotoxicity assessment using alkaline single cell gel electrophoresis (comet) assay, as previously established [53]. Blood samples were quickly mixed with low melting point agarose (100 µL 0.75% in PBS, Invitrogen, Waltham, MA, USA) and spread onto slides pre-coated with normal point agarose (1.5% in PBS, Invitrogen, Waltham, MA, USA) and covered with coverslips. Following agarose solidification (4 • C for 5 min), coverslips were carefully removed. The slides were incubated with cold lysis solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-HCl, 1% N-lauroyl-sarcosine, 1% Triton X-100 and 10% DMSO, pH 10) overnight, at 4 • C. Subsequently. The slides were washed three times in PBS and immersed in a fresh cold alkaline electrophoresis buffer (300 mM NaOH, 1 mM Na 2 EDTA, pH > 13) for 20 min. Electrophoresis was conducted for 20 min at 1 V/cm (300 mA) for 20 min. The slides were then neutralized with 0.4 M Tris (pH 7.5), dehydrated in 100% ethanol, and stained with SYBR Gold solution (1:10,000) (Invitrogen, USA). Fifty randomly selected nucleoids were scored in each slide (two slides/animal) under an epi-fluorescence microscope (Olympus BX-50, Shinjuku, Japan) using Comet Assay IV software (Perceptive Instruments, Great Shelford, UK). Tail intensity (% of DNA in comet tail) and Tail moment (% of DNA in comet tail/tail length) was chosen as reliable parameter to evaluate DNA damage [54].

ALT, Creatinine and Urea Serum Determination
Serum samples (−20 • C) were used to evaluate alanine aminotransferase (ALT) enzyme levels (i.e., hepatic damage), creatinine and urea (i.e., kidney function) using the colorimetric enzymatic method with the use of commercial kits (BioClin, Belo Horizonte, Brasil) in an automatic spectrophotometric analyzer (Chemistry Analyzer Bs-200, Mindray Medical International Limited, Shenzhen, China).

Histopathological Evaluation and Collagen Morphometry
Formalin-fixed liver and kidney samples were embedded in paraffin blocks, and 5 µm sections were obtained and stained with hematoxylin and eosin (HE) or Sirius Red. Histopathological alterations in both organs were identified in a section stained with HE, following the criteria established in the literature [23,55]. The incidence (% lesionbearing rats/group) was evaluated in all experimental groups. Collagen morphometry was analyzed in Sirius red-stained sections. Kidney (cortex and medulla) and liver (portal areas and Glisson's capsule) were randomly photographed (40× objective, 10 fields/slide/rat), and the collagen area was calculated using Leica QWin 3.0 software (Leica Microsystems, Wetzlar, Germany).