L-Carnitine alleviates hepatic and renal mitochondrial-dependent apoptotic progression induced by letrozole in female rats through modulation of Nrf-2, Cyt c and CASP-3 signaling

Abstract Letrozole (LTZ) is a non-steroidal aromatase inhibitor that is commonly used in breast cancer therapy. It has several side effects that might lead to the drug's cessation and data of LTZ's potential adverse effects on the hepatorenal microenvironment was conflicting. In addition, searching for therapeutic interventions that could modulate its adverse effects will be very beneficial. So, this study aims to determine the impact of LTZ on the hepatorenal microenvironment in cyclic female rats with a proposed regulatory role of L-Carnitine (LC) supplementation giving molecular insights into its possible mechanism of action. LTZ (1 mg/kg using 0.5% carboxy methyl cellulose as a vehicle for 21 consecutive days orally) to assess its impact on hepatorenal microenvironment. After treatment with LC (100 mg/kg orally) for 14 days, hepatorenal redox state (lipid peroxides (MDA), reduced glutathione (GSH) and catalase enzyme (CAT)), as well as relative gene expression of nuclear factor erythroid 2-related factor 2 (Nrf-2), cytochrome-c (Cyt c) and caspase-3 (CASP-3) were evaluated. Histopathological examination and immunohistochemical staining of CASP-3 in both liver and kidney were done. LTZ altered hepatic and renal functions. Relative gene expression of hepatorenal Nrf-2, Cyt c and CASP-3 as well as redox state revealed significant deterioration. Also, the liver and kidney tissues showed several micromorphological changes and intense reaction to CASP-3 upon immunohistochemical staining. It can be concluded that LC alleviates LTZ induced hepatorenal oxidative stress (OS) and mitochondrial-dependent apoptotic progression through modulation of Nrf-2, Cyt c, and CASP-3 signaling in female rats.


Introduction
Breast cancer is the leading cause of death among women around the world (Thiantanawat et al. 2003). Evidence of an increasing burden of both premenopausal and postmenopausal breast cancer has emerged worldwide (Heer et al. 2020). Estrogen deprivation can operate as a therapeutic target for cancer treatment since estrogens are required for estrogen receptor-positive tumor development (Rabaglio and Ruepp 2010). Aromatase inhibitors (AIs) are commonly used in the treatment of breast cancer (Thiantanawat et al. 2003). AIs suppress the circulating level of estrogens by blocking the aromatase enzyme, which reduces estrogen synthesis (Zych et al. 2018). Letrozole (LTZ) is one of the third-generation AIs that is widely used in breast cancer therapy (Aydin et al. 2011). Also, LTZ can be used as a fertility regulating agent in women and animal models (Kilic-Okman et al. 2003, Aydin et al. 2011. However, AIs are associated with several relevant side effects that have been reported in 30%-60% of treated patients, which can lead to its use being restricted and even drug discontinuation. (Fusi et al. 2014). Among these side effects is the AI-associated negative impact on the level of both liver and kidney (Zapata et al. 2006, Kalender et al. 2007, Aydin et al. 2011, Boutas et al. 2015, Gharia et al. 2017, Puri et al. 2020. Data on the impact of LTZ on the hepatorenal microenvironment using various animal models is highly debatable and controversial (Galal et al. 2018, Chaiyamoon et al. 2020. Furthermore, there is a scarcity of data on the effect of LTZ on hepatic and renal tissues in female rats during reproductive age. As women of reproductive age are particularly vulnerable to the use of hepatic injury drug triggers (Rabaglio and Ruepp 2010). The deleterious effects of AIs in this population, as well as their probable mechanisms, are poorly understood. Antiestrogens and AIs impede cell cycle progression and activate apoptosis (Thiantanawat et al. 2003). As a result, it seemed relevant to investigate the effect of LTZ on hepatic and renal tissues in female rats during reproductive age employing biochemical parameters, oxidative stress (OS), apoptotic markers, and histopathological investigations. Consequently, we can pinpoint LTZ's potential side effects, gaining molecular insight into its mechanism of action and exploring for therapeutic approaches to mitigate its negative consequences.
L-Carnitine (LC) acts as a mitochondrial regulatory nutrient that targets OS/antioxidant imbalance (Ranjbar Kohan et al. 2020). LC is essential for ATP production by mitochondrial b-oxidation of fatty acids (Agarwal et al. 2018, Aboubakr et al. 2020. Also, LC could significantly regulate OS and apoptosis in different cell types (Elkomy et al. 2020). Therefore, the main goal of this study is to see how LTZ alters the hepatorenal microenvironment in cyclic female rats, as well as the potential regulatory role of LC supplementation.

Animal care and ethical approval
All animal procedures and experimental details were under approval and authorization of the Institutional Animal Care and Use Committee (IACUC), Cairo University, Egypt (CUIIF921). Female Sprague-Dawley rats (4-6 weeks old) were housed in the Laboratory animal house of the Physiology department, Faculty of Veterinary Medicine, Cairo University. Animals were housed with a 12 hrs light-dark cycle at 22 ± 2 C and 55%-65% humidity. All animals had free access to water and standard chow. Animals were accommodated with laboratory conditions for at least 2 weeks before treatment and maintained under the same conditions all over the experiment.

Experimental design
In the current experiment, 24 female albino rats of average bodyweight 200-250 gm showing 4-5 days normal estrus cycle were selected and randomly divided into four groups (n ¼ 6). Group (1) served as a control and was orally administrated by oral gavage with 0.5% aqueous solution of carboxymethylcellulose (CMC). Group (2) was orally gavaged with LTZ at the concentration of 1 mg/kg dissolved in 0.5% CMC for 21 days and allowed for 14 days recovery period. Group (3) was orally given LTZ (1 mg/kg) dissolved in 0.5% CMC) for 21 days followed by LC that was orally gavaged at dose of (100 mg/kg) for 14 days, Group (4) was orally given 0.5% aqueous solution of CMC for 21 days followed by oral administration of LC (100 mg/kg) for 14 days. All applications were carried out daily, and the experiment was extended for 35 days. Vaginal smears were taken daily to assess estrous cyclicity. Eventually, animals were anesthetized on the 35th day of the experiment, and blood samples were collected, then animals were decapitated, and organs (liver and kidney) were collected for histopathological examination and OS and gene expression assessment.

Dose and gender selection
Letrozole oral dose (1 mg/kg) was previously reported to be hepatotoxic in female rats (Aydin et al. 2011). LTZ dose was obtained from previous studies (Kumru et al. 2007, Aydin et al. 2008, Taylor et al. 2017. LTZ has 99.9% bioavailability upon oral administration (Yang et al. 2021). LC oral dose (100 mg/kg) was shown to have hepatic and renal antioxidant as well as antiapoptotic potential (Elkomy et al. 2020).
There is a lack of information on the effect of LTZ on hepatic and renal tissues in female rats during reproductive age. Premenopausal females receiving endocrine treatment for early breast cancer are among the age groups at risk for taking medicines or other substances that cause liver damage. There is little known regarding the side effects of AIs at the level both liver and kidney in cyclic females (Rabaglio and Ruepp 2010). In addition, LTZ is widely used in the field of female infertility (Yang et al. 2021). Therefore, adult cyclic female rats were chosen in our study.

Cage side observations, morbidity and mortality
Changes in fur, eyes, and mucous membranes, as well as respiratory pattern, gait, posture and fecal aspects, were all routinely observed inside the cages daily. All animal cages were checked on a daily basis for morbidity and mortality.

Body weight gain and feed intake
Food consumption was tracked on a daily basis. The feed intake was calculated by subtracting the amount of feed left in the feeder from the initial known amount, assuming that all of the animals "wasted" roughly the same amount of feed. Using an electronic weighing balance, the gain in body weight of each rat in each group was calculated on a weekly basis during the study period to determine the effect of treatments on body weight. By subtracting the initial weight from the final weight, the overall weight gain was estimated.

Blood and tissue sampling
Individual fasting blood samples (for 10 hrs.) were collected at 8 am from rats of all experimental groups at the 5 th week. Blood was collected in specific tubes for serum separation then left to stand for 15 mins and finally centrifuged at 4000 rpm for 10 mins. Serum samples were collected and stored at À20 C for further biochemical analysis and hormonal assays. Before dissection, liver and kidney tissues were washed with phosphate-buffered (PH 7.4) saline (PBS) containing 0.16 mg/ml heparin to remove RBCs and clots, then they were weighed and dissected into two parts. The first part was stored at À80 C for the determination of antioxidant status and RT-PCR analysis, while the other part was fixed in 10% neutral buffered formalin (10% NBF) and processed for histological and immunohistochemical investigation.

Oxidative stress/antioxidant assessment
Hepatic and renal tissues were weighed and then homogenized with cold phosphate buffer (pH 7.4) and centrifuged at 4000 rpm for 15 mins at 4 C. The collected supernatant was used to determine the OS/antioxidant parameters of the following assays: CAT (catalase assay) was assessed (Aebi 1984). Reduced glutathione (GSH) was estimated by the method as described previously (Beutler et al. 1963). Malondialdehyde (MDA) was measured (Ohkawa et al. 1979). All parameters were detected using special kits purchased from Bio-diagnostic Company, Egypt.

Serum biochemical analysis
The activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined in collected sera as markers for liver injury (Reitman and Frankel 1957), while albumin and total protein were assessed as markers for liver function (Doumas et al. 1972, Doumas et al. 1981. In addition, serum levels of urea, uric acid, and creatinine were measured to evaluate kidney function (Coulombe and L 1963, Bartels et al. 1972, Tiffany et al. 1972).

RT-PCR analysis
qRT-PCR analysis for Nrf-2, cyt c and CASP-3 genes The relative hepatic and renal Nrf2, Cyt c, and CASP-3 mRNA abundance were determined by qRT-PCR of cDNA samples, using GAPDH as a housekeeping gene. Approximately, 100 mg of hepatic and renal tissues were used for total RNA extraction using the total RNA Extraction Kit (Vivantis, Malaysia). After confirming the concentration and purity of RNA, RT-PCR was performed using M-MuLV Reverse Transcriptase (NEB#M0253). Quantitative assessment of DNA amplification for each gene was performed by a fluorescence-based real-time detection method with a fluorescent SYBR Green dye (Thermo Scientific, Cat. No. K0221). The realtime PCR conditions were performed as follows: 95 C for 5 mins (initial denaturation) and then 40 cycles at 95 C for 15 s, 60 C for 30 s, and 72 C for 30 s in each experiment. Negative controls that were free of the template were included. Each qRT-PCR was performed with three biological replicates, and each biological replicate was assessed three times. The comparative 2 ÀDDCT method was used to calculate the relative transcription levels (Livak and Schmittgen 2001) (Table 1).

Immuno-histochemical examination for caspase 3
Immunohistochemical study for detection of the Caspase-3(CASP-3) was performed on paraffin sections and mounted on positively charged slides. CASP-3 was used as a marker for apoptosis according to Hsu et al. (1981).

Evaluation of caspase 3 immunohistochemistry using (area %)
Immunohistochemically stained sections for liver and kidney tissues were examined using Leica Qwin 500 analyzer computer system (Leica Microsystems, Switzerland) in the Faculty of Veterinary medicine, Cairo University. Liver and kidney tissues for CASP-3 immunostaining were measured as area % in a standard measuring frame in (5 fields/group) using magnification (x400) by light microscopy transferred to the screen. The areas showing brown immunostaining were considered as CASP-3 positive. These areas were chosen for evaluation, regardless the intensity of staining. Mean values and standard deviations were obtained for each specimen.

Statistical analysis
Statistical analysis was performed using SPSS (Version 20.0; SPSS Inc., Chicago, IL, USA) including the calculations of Means ± standard deviations (SD) using one-way analysis of variance (ANOVA) accompanied by the Duncan test as a post hoc. Significance was set at the p < 0.05 level. Table 1. Primer sequence used for qRT-PCR.

Results
Cage side observations, morbidity and mortality

Body weight gain and feed consumption
Supplementary Table 2. Showed that there were no significant differences in body weight gain between different groups at p < 0.05. Also, average daily feed intake in female rats showed no significant alteration throughout experimental period between different groups at p < 0.05. Table 2 demonstrated that both AST and ALT activities in the LTZ group were significantly increased at p < 0.05 when compared to the control group. LC supplementation for 14 days resulted in a significant decrease in liver enzymes activities when compared to the LTZ group at p < 0.05. Furthermore, when comparing the LTZ exposed group to the control group at p < 0.05 total protein and albumin serum levels exhibited no significant differences. LC treatment for 14 days showed no significant difference in serum total protein as well as albumin levels between the LTZ group and even the control group at p < 0.05. Also, urea, uric acid and creatinine concentrations were significantly elevated at p < 0.05 in the LTZ treated group in comparison with the control group, and upon treatment with LC, urea, uric acid, and creatinine levels showed significant improvement.

Oxidative stress/antioxidant assessment
Oxidative stress (OS)/antioxidant assessment in both hepatic and renal tissues was illustrated in Figure 1. LTZ administration for 3 weeks showed a significant decline at p < 0.05 in hepatic and renal reduced GSH content as well as CAT enzyme activity. MDA level was significantly elevated at p < 0.05 compared to the control group. LC supplementation showed significant improvement at p < 0.05 of both hepatic and renal tissue GSH levels as well as CAT activity when compared to the LTZ group. Hepatic and renal MDA levels were significantly inhibited at p < 0.05 upon LC treatment, in comparison with the LTZ group.

RT-PCR analysis
Data illustrated in Figure 2 revealed that Nrf-2 gene was significantly downregulated at p < 0.05 in the LTZ group, compared with the control one in both liver and kidney. On the other hand, the expression of Cyt c and CASP-3 genes were significantly upregulated in the LTZ group (p < 0.05), compared with the control one in both the liver and kidney samples. LC treatment significantly increased the expression level of Nrf-2 gene from 0.41 to 0.75-fold in the liver, and from 0.1 to 0.4-fold in the kidney (p < 0.05), when compared with the LTZ group. LC administration also modulated the expression level of Cyt c and CASP-3 genes from 4 to 1.5-fold, and from 2.78 to 1.4-fold in the liver (p < 0.05) respectively, when compared to the LTZ group. On the same line the two genes were significantly downregulated in the kidney from 5 to 1.4fold and from 7.2 to 2.5-fold (p < 0.05) respectively compared with the LTZ group.

Histopathological findings of hepatic tissue sections stained by H&E
Microscopical examination of H&E-stained sections of hepatic tissue of the control group revealed normal histological architecture. Meanwhile, LTZ exposed group showed distortion of the radiating hepatic cords, congestion, and dilatation of the central vein, in addition to vacuolation and degeneration of hepatocytes. Blood sinusoids appeared dilated with active Von Kupffer cells. The hepatic tissue of rats treated with LC exhibited normal homogenous hepatic parenchyma. However, some hepatocytes showed cytoplasmic vacuolation, and inflammatory cells were seen aggregated in some parts of the hepatic parenchyma. The hepatic tissue of LTZ group treated with LC exhibited normal polyhedral hepatocytes with large spherical central and lightly stained nuclei. Less congestion of the central vein and blood sinusoids was noticed LTZ group treated with LC when compared with the group exposed to LTZ alone. In addition, few patches of inflammatory cell aggregations were also noticed in the hepatic parenchyma of LTZ group treated with LC ( Figure 3 and Table 3).

Histopathological findings of renal tissue sections stained by H&E
On the other hand, examination of the control group showed normal renal tissue. LTZ exposed group showed distorted renal glomeruli with narrowing and closure of the glomerular space. In addition, the renal medulla exhibited protein casts in the renal tubules which showed necrosis of their epithelial lining, and some of them appeared desquamated. Severe congestion and edema were also noticed in the renal blood vessels. Meanwhile, LC exposed group revealed normal renal corpuscles and renal tubules with intact epithelial lining. However, few corpuscles showed narrowing of the glomerular space and few renal tubules exhibited cytoplasmic vacuolation. The kidneys of rats in LTZ group treated with LC revealed fewer alterations in the renal parenchyma when compared with the group treated with LTZ alone. Also, LTZ group treated with LC exhibited less distorted renal glomeruli; most of them appeared normally, while few of them showed narrowed glomerular space. The renal tubules in LTZ group treated with LC appeared with intact lining epithelium, but some of them showed cytoplasmic vacuolation which was less than what was appeared in LTZ exposed group (Figure 4 and Table 4).

Immunohistochemical for caspase 3 (CASP-3)
Immunohistochemical examination of CASP-3 for hepatic and renal tissues revealed a strong immunoreactivity in the LTZ group when compared with the control group, while LTZ group treated with LC showed improvement in CASP-3 immunoreactivity in hepatorenal tissue. Figures 5 and 6, and Table 5.

Discussion
The current study showed that LTZ administration in female rats resulted in a significant elevation in liver and kidney functions. Similar results were obtained by Aydin et al. reported altered hepatic MDA and GSH reductase activity in LTZ PCOS animal models. Inconsistency with our results, Galal et al. (2018) found significant upregulation in the expression of genes related to renal OS in ovariectomized rats treated with AIs. Reactive oxygen species (ROS) act as dual-edged swords; as they play a critical role in several physiological systems, but under conditions of oxidative stress, they contribute to cellular dysfunction and even injury (Roberts and Sindhu 2009). OS results in macro-molecular damage to carbohydrates, proteins, lipids, and nucleic acids (Gyur aszov a et al. 2020). We thought that LTZ induced cellular estrogen deprivation also could be a major player in the pathogenesis of AIs associated liver and kidney adverse effects. Estrogen deficiency leads to excessive mitochondrial and peroxisomal ROS generation and depletes hepatocellular GSH and elevates MDA (Cristine de Oliveira et al. 2018). Under both physiological and pathological conditions, estrogen and its primary metabolites have been shown to have reno-protective effects (Mankhey et al. 2005). Thus, estrogen deficiency exaggerates OS induced kidney damage resulting from the imbalance between ROS and their neutralization by endogenous antioxidant defense mechanisms (Mankhey et al. 2005). However, further investigations are needed to prove that.
Oxidative stress (OS) can induce cellular damage via modulation of the pathways that control expression of genes and predispose to apoptosis via modulation of signals that control the expression of genes regulating programmed cell death (Li et al. 2015). Our results were confirmed by liver and kidney micromorphological deteriorations. In the current study, the compromised antioxidant defense system in the LTZ group was evident in the form of depressed expression of nuclear factor (erythroid-derived 2)like 2 (Nrf-2) gene in both hepatic and renal tissues. Nrf-2 gene is a master regulator of a battery of antioxidant and detoxification genes with a cytoprotective function (Tonelli et al. 2018). When Nrf-2 is exposed to OS, it phosphorylates and gets translocated into the nucleus enhancing the transcription of proteins and antioxidant enzymes (Li and Kong 2009). In addition, Cyt c is one of the major actors in the apoptotic scene and acts as a quiet worker on the respiratory chain. It can get released from the cell's energy house, the mitochondrion, when this organelle is damaged or when it receives instructions for its outer membrane breakdown and can be recruited into the apoptotic dismantling of the cell (Garrido et al. 2006). However, mitochondrial   . Therefore, administration of LTZ led to significant upregulation of Cyt c gene expression due to hepatorenal redox state imbalance and predisposition to a mitochondrial dependent apoptosis. As, Cyt c is the master regulator gene encoding the transcriptional factor that is implicated in the regulation of apoptosis, cell proliferation, and transformation (Rashad et al. 2018). Cyt c activation leads to the activation of CASP 3, which is considered the main executor of apoptosis and chromatin condensation (Hotti et al. 2000). Therefore, LTZ administration led to upregulation in the gene expression of CASP-3 (an apoptotic marker) and was confirmed by an exaggerated immune reaction to it upon immunohistochemical staining of both hepatic and renal tissues. CASP activation is one of the commitment steps leading to apoptosis that can be triggered through different stimulatory pathways (Elmore 2007). Also, CASP-3 was reported to be activated in the estrogen-deprived cells (Monroe et al. 2002). This could explain our reported data that confirm significant histological alterations at the level of both liver and kidney tissues due to activation of apoptosis as a result of oxidative damage induced by LTZ.
Drug revolution nowadays is promising through providing several antioxidant supplements that afford favorable regulatory pathways. LC has been demonstrated to improve membrane stability through its potent antioxidant potential (Evangeliou andVlassopoulos 2003, Bene et al. 2018). Therefore, it can protect against OS induced in various tissues, including the liver and kidney (Cayir et al. 2009). In line with our results, LC showed significant improvement in liver and kidney biochemical parameters as well as protection against hepatorenal OS and mitochondrial induced apoptosis imposed by LTZ administration. Our results were confirmed by the results of histopathology as significant improvement in hepatic and renal architectures was noticed upon LC administration. The ability of LC to significantly improve the liver and kidney architectures may be due to its antioxidant and anti-apoptotic effects and its capability to act as a free radical scavenger, leading to the protection of membrane permeability (Augustyniak and Skrzydlewska 2009). In the current study, the antioxidant and anti-apoptotic effects of LC were represented in the upregulation of Nrf-2 and downregulation of Cyt c and CASP-3 genes. It has been reported that the hepatoprotective effect of LC was associated with Renal tubules showed -protein casts À 0/5 þ 2/5 À 0/5 À 0/5 -necrosis À 0/5 þ 1/5 À 0/5 À 0/5 -desquamation À 0/5 þ 2/5 À 0/5 À 0/5 Congestion of renal vessels À 0/5 þþþ 4/5 þþ 3/5 À 0/5 Inflammatory cell infiltration in renal tissue  Nrf-2 signaling pathway that is implicated in the regulation of apoptosis (Li et al. 2016). In addition, LC restored oxidative balance in renal tissue and antagonized CASP-3 mediated apoptotic cell death (Kunak et al. 2016). Also, Nrf-2 has a significant role in protecting the kidney from oxidative damage (Nezu and Suzuki 2020). LC supplementation suppresses CASP-3 activity, enhances total antioxidant capacity and scavenges free radicals (Kelek et al. 2019). Our results were strengthened by immunohistochemical findings, as the percent area covered by CASP-3 positive immunoreactive cells within the hepatic and renal tissues was significantly inhibited upon LC treatment. Since CASP-3 can be activated by ROS, the suppressive effect of LC on CASP-3 activity suggests that the inhibitory effect could also be related to the antioxidant property and mitochondrial protection of LC (Kelek et al. 2019). LC supplementation can be potentially used to lessen both oxidative and apoptotic progression in hepatic and renal tissues, as it was reported to regulate apoptotic protein expression and to suppress CASP-3 activity (Kelek et al. 2019).

Conclusion
It can be concluded that LTZ administration for 21 days in female rats is associated with deterioration of hepatorenal redox state, significant alteration in the genes regulating OS and apoptosis in liver and kidney tissues. LC supplementation for two weeks significantly mitigated hepatorenal OS and mitochondrial-dependent apoptosis imposed by LTZ. LC exerts its action through its antioxidant and antiapoptotic properties.

Disclosure statement
The authors declare no conflicts of interests exist relevant to the content of this article.