3-Indolepropionic Acid Prevented Chlorpyrifos-Induced Hepatorenal Toxicities in Rats By Improving Anti-Inammatory, Antioxidant And Apoptotic Responses and Abating DNA Damage

We examined the individual and combined effect of 3-Indolepropionic acid (IPA) and Chlorpyrifos (CPF) on rat hepatorenal function. The experimental cohorts (n=6) were treated per os for 14 consecutive days as follows: Control (Corn oil 2 mL/kg body weight), CPF alone (5 mg/kg), IPA alone (50 mg/kg) and the co-treated cohorts (CPF: 5 mg/kg + IPA: 25 or 50 mg/kg). Biomarkers of hepatorenal damage, antioxidant and myeloperoxidase (MPO) activities, the levels of nitric oxide (NO), lipid peroxidation (LPO) and reactive oxygen and nitrogen (RONS) species were spectrophotometrically evaluated. Besides, the concentration of tumour necrosis factor-alpha (TNF- α), interleukin-1 β (IL-1β) and caspase-3 activity and 8-hydroxy-2’-deoxyguanosine adducts (8-OHdG) was also assessed by Enzyme-Linked Immunosorbent Assay. Treatment with CPF alone increased biomarkers of hepatorenal toxicity was signicantly (p<0.05) alleviated in rats co-exposed to CPF and IPA. Moreover, the decrease in antioxidant status as antioxidant elevation in RONS and LPO were lessened (p<0.05) in rats co-treated with CPF and IPA. CPF mediated increases in TNF-α, IL-1β, NO, MPO and caspase-3 activity were reduced (p<0.05) in the liver and kidney of rats co-exposed to CPF and IPA. In addition, 8-OHdG adducts formation were reduced in rats treated with 3-IPA dose-dependently. Light microscopic examination showed that histopathological lesions severity induced by CPF were alleviated in rats co-exposed to IPA and CPF. In conclusion, the results demonstrated that rats co-exposed to IPA and CPF exhibited reduced CPF-induced oxidative stress, inammation, DNA damage and caspase-3 activation of the liver and kidney.


Introduction
Chlorpyrifos (CPF) is a broad-spectrum organophosphate compound mainly used as a pesticide to control soil-borne insects and mites and as a herbicide to control weeds and foliage (Pal, Kokushi, Koyama, Uno, & Ghosh, 2012). CPF exposure occurs primarily through consuming contaminated foods, inhalation, and absorption through the skin during preparation and application (Albasher, Almeer, Alari , et al., 2019). As a result, the risk of exposure and uncontrolled use of organophosphate (OP) insecticides such as CPF has increased worldwide, especially in developing countries (Kokushi, Uno, Pal, & Koyama, 1964;Yu, Kim, & Kang, 2011). Over the years, IPA has been established as a unique potent antioxidant devoid of pro-oxidant activity (Bendheim et al., 2002). Contrary to other antioxidants, IPA is not converted to reactive intermediates with pro-oxidant activity and does not undergo autoxidation in the presence of transition (Bendheim et al., 2002). As a result, IPA has been shown to mitigate lipid peroxidation, carcinogenesis, accumulation of 8-OH-dG and subsequent DNA damage induced by several compounds such as chromium, 17β-Estradiol (E 2 ), Iron due to its hydroxyl radical scavenging ability (Aust & Eveleigh, 1999; M. Karbownik To our knowledge, no such study is currently present in the scienti c literature. We treated rats with CPF and IPA for four consecutive weeks and evaluated hepatic and renal toxic responses to achieve these goals. Speci cally, we assessed serum level of hepatorenal toxicity, biomarkers of in ammatory responses, oxidative stress, apoptosis besides a biomarker of DNA damage and histopathological changes in experimental rats hepatorenal system. The data we report here present more insight into IPA supplementation's bene cial effect and the underlying mechanisms of IPA protective activity. We conclude that 3-IPA dose-dependently augmented the antioxidative capacity of rats, decreased oxidative stress, apoptosis, and in ammatory responses. IPA also prevented histological damages in the liver and kidney of rats resulting from CPF toxicity.

Animal care and experimental design
For the avoidance of type I and II errors, sample size computation was done using the G* Power software version 3.1.9.4 (Faul, Erdfelder, Lang, & Buchner, 2007). The effect size of 0.40 (Larger effect) at .05 alpha error of probability for one way analysis of variance (ANOVA) was adopted in the study, and this concurs with the Cohen's guideline (Cohen, 1992). Based on this, a total sample size of 125 was obtained at 95% power. To adhere to the 3R guidelines for the welfare and use of experimental animals ( for this study. The experimental rats were acclimatised for seven days before they were subjected to different treatments. The rats were fed with standard rat pellets (Ladokun Feeds Limited) and water ad libitum and subjected to natural photoperiod of about 12 hours of light/darkness cycle daily. The present study consisted of ve experimental groups, with each group containing six rats each. They were treated for 14 consecutive days. Group I (Control) rats received normal drinking water, Group II (CPF alone) rats orally received 5 mg/kg body weight, Group III (1PA alone) rats received IPA alone in 50 mg/kg. In contrast, Group IV (CPF + IPA) rats received CPF and IPA1 25 mg/ kg and Group V received CPF and IPA2 50 mg/ kg. Separate stock solutions (5 mg/kg) of CPF and IPA were prepared freshly every other day. The doses of CPF (5 mg/kg) and IPA (50 mg/kg) used in the current study are selected based on previously published data (Owumi &

Termination of the experiment and excision of tissues
Twenty-four hours after the last treatment, blood was collected into non-heparinised tubes and the rats sacri ced by cervical dislocation after carbon dioxide (CO 2 ) asphyxiation (AVMA, 2001; Hawkins et al., 2016; Owumi, Nwozo, Arunsi, Oyelere, & Odunola, 2021). Subsequently, the serum was prepared by centrifuging the clotted blood at 4,000g for 10 minutes using a centrifuge. The liver and kidney were excised, rinsed in ice-cold 1.15% KCl solution, blotted with lter paper and weighed. The organs were sectioned for histological examination, and the remaining portion homogenized in 0.1M of phosphate buffer (pH 7.4) using a Te on homogeniser. The resulting homogenates were centrifuged at 12,000 rpm for 15 minutes using a cold centrifuge to obtain the post mitochondria fraction. Finally, the supernatant was collected and used for biochemical analyses.

Evaluation of liver and kidney function indices
Liver function indices, namely the serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP),gamma-glutamyl transferase ( -GT), were evaluated using available commercial kits from Randox TM Laboratories Limited, UK. Moreover, kidney functions indices, namely serum urea and creatinine concentrations, were determined using Randox TM Laboratories Limited (Crumlin, UK).
Additionally, TRX and TRXR levels were evaluated using commercially available ELISA Kits (Elabscience Biotechnology Company, Beijing, China) with the aid of a SpectraMax plate reader (Molecular Devices, CA, USA) as stated in the manufacturer's manual. following the manufacturer's protocol. All reading was obtained using an M384 SpectraMax TM Multimodal plate reader (Molecular Devices, San Jose, CA, USA).

Assay of apoptotic and DNA damage biomarkers
Caspase-3 and caspase-9 activities and 8-OHdG level were evaluated using commercially available ELISA Kits (Elabscience Biotechnology Company, Beijing, China) with a SpectraMax plate reader (Molecular Devices, CA, USA) as stated in the manufacturer's manual.
Histological evaluation of the liver and kidney tissues Liver and kidney samples that were earlier xed in phosphate-buffered formalin (10%; for three days) were randomly selected for histological analysis. The samples were embedded in para n following sequential dehydration processes. Subsequently, microtome sliced tissue-liver and kidney-(4-5µm) were xed on charged microscopic glass slides and stained with hematoxylin and eosin (H & E) following established protocol (Bancroft & Gamble, 2008; Owumi, Olusola, Arunsi, & Oyelere, 2021). Histopathologic examination of hepatic and renal tissue was conducted using a Carl Zeiss Axio-Scope A1 light microscope (G ttingen, Germany) to identify pathological abnormalities. The slides holding the tissue were scored by a pathologist oblivious of the experimental groups. Representative images were captured from the slides using a Carl Zeiss Axiocam 105 digital colour Camera (G ttingen, Germany) attached to the microscope.

Statistical Analysis of Results
The analysis of the data generated from this study was performed by one-way analysis of variance (ANOVA) followed by a post-hoc test (Bonferroni) using GraphPad Prism version 8.3.0 for Mac (www.graphpad.com; GraphPad, CA, USA,). Statistically signi cant differences were set at values of p<0.05. The results are expressed as the mean ± SD of replicates.

Effects of CPF and IPA on organ weight and markers of hepatorenal injury in rats
There is a signi cant (p < 0.05) reduction in the liver of rats treated with CPF alone compared to control by 7.37% Table 1. The kidney of animals treated with CPF alone showed no difference in weight relative to control. The kidney of rats co-treated with CPF and IPA (25 and 50 mg/kg) increased by 2.76%, while the liver of rats co-treated with CPF and IPA (25 and 50 mg/kg) increased by 10.29% and decreased by 5.98% compared to CPF alone treated rats. The effect of IPA on CPF-induced hepatic and renal toxicities was evaluated from serum levels of hepatic transaminases and creatinine and urea, respectively Fig. 1. Exposure to CPF (5mg/kg) increased (p < 0.05) biomarkers of hepatic and renal injury in rat serum compared to the control rats. The activities of AST, ALT, ALP, and GGT and creatinine and urea levels increased by 13.28%, 12.57%, 82%, 86.18%, 60.41%, and 76.49%, respectively when compared to the control group. In rats co-treated with IPA (25 and 50 mg/kg) and CPF, IPA decreased CPF-mediated increases by (76.53%, 72.70%) in the level of urea. Rats co-treated with IPA and CPF (25 mg/kg) showed no decrease in creatinine level. However, rats co-treated with IPA and CPF (50 mg/kg) showed a signi cant reduction in creatinine level by 6.2% compared to CPF alone treated animals. Furthermore, a signi cant decrease in the serum activity of ALT by (29.82% and 1.67%), AST by (99.43% and 83.56%), ALP by (24.66% and 4.04%) and GGT by (122.64% and 124.17%) in rats co-treated with 3-IPA (25 and 50 mg/kg) compared to rats treated with CPF alone.  25 and 50 mg/kg) in rat kidney co-treated with IPA and CPF. Also, exposure to CPF alone markedly decreased GSH in the kidney and liver of the treated rats when compared to the control group Fig. 3. The level of GSH reduced signi cantly by 61.9% in the liver and 61.54% in the kidney of rats treated with CPF alone compared to the control group. The level of GSH in the co-treated groups increased by 49.35% (IPA1) and 79.17% (IPA2) in the liver and by 56% (IPA1) and 28.57% (IPA2) in the kidney when compared to rats treated with CPF alone.
Effect of CPF and IPA on hepatic and renal RONS generation and induction of LPO and total thiol levels in rats The effect of IPA on RONS generation and subsequent LPO-induction in CPF treated rats were evaluated, and the results are shown in Fig. 4. Exposure to CPF alone evidently increased (p < 0.05) RONS levels in the kidney by 107.5% and liver by 141.5% compared with the control group. LPO levels in the kidney and liver of the CPF treated rats also signi cantly increased by 80% and 92.71%, respectively, compared with the control group. Co-treatment with IPA protected against renal and hepatic damage evidenced by decreases (p < 0.05) in RONS and LPO levels than rats treated with CPF alone. There was a reduction in RONS levels in groups co-treated with CPF + IPA1 and CPF + IPA2 by 59.7% and 108.6%, respectively, compared to CPF alone in the liver 0.87% and 36.17% respectively compared to CPF alone group in the kidney. LPO in groups co-treated with CPF + IPA1 and CPF + IPA2 was reduced (p < 0.05) by 95.33% and 132.87% compared to CPF alone treated groups in the liver and by 50.82% and 49.45% in the kidney when compared to CPF alone treated rats. Also, TSH levels decreased (p < 0.05) in the liver and kidney of rats treated with CPF alone by 58.21% and 71%, respectively, compared to the control group. TSH levels in the co-treated groups increased 56.25% (IPA1) and 79.37% (IPA2) in the liver, and 76.09% (IPA1) and 98.22% (IPA2) in the kidney compared to rats treated with CPF alone.

Effects of CPF and IPA treatments on biomarkers of in ammation in rats
The in uence of IPA on the biomarkers of in ammation was assessed in the liver and kidney of CPFtreated rats. Administration of CPF increased (p < 0.05) the hepatic and renal MPO, XO activity, and level of NO compared to the control group Fig. 5. In the liver, the activities of MPO and XO in CPF alone treated rats increased by 7.74% and 158.85%, respectively, compared to the control group; MPO and XO levels in the kidney also increased 58.58% and 90.32% compared to the control. Results also show an increase in the kidney and liver levels of NO by 47.16% and 3.77%, respectively, compared to control groups. However, administration of 3-IPA abrogated CPF-induced increase in these biomarkers of in ammation in the liver and kidney of rats compared with rats administered CPF alone. MPO activities in groups co-treated with CPF + 3-IPA1 and CPF + 3-IPA2 were reduced (p < 0.05) by 49.53% and 34.29% compared to CPF alone treated groups liver by 37.22% and 3.43% in the kidney when compared to CPF alone treated rats. There was a reduction in XO activities in groups co-treated with CPF + IPA1 and CPF + IPA2 by 90.70% and 168.55%, respectively, compared to CPF alone in the liver and 121.43% and 47.46% respectively compared to CPF alone group in the kidney. There was also a reduction in the levels of NO in groups cotreated with CPF + IPA1 and CPF + IPA2 by 12.59% and 43.58%, respectively, compared to CPF alone the liver and 1.12% and 27.87% respectively compared to CPF alone group in the kidney.
Effects of CPF and IPA on biomarkers of apoptosis in rats Caspase − 3 and − 9 activities increased (p < 0.05) in the liver by 116.8% and 133.3% and in the kidney by 92.7% and 97.7%, respectively, compared to the control group Fig. 7. Co-treatment with IPA protected the liver and kidney from excessive apoptosis in a dose-dependent manner, as seen in the signi cant decreases (p < 0.05) in caspase-3 and caspase-9 activities compared with rats treated with CPF alone. There was a reduction in caspase 3 activity in groups co-treated with CPF + IPA1 (69%; 34.8%) and CPF + IPA2 (90.2%; 64.2%) in the liver and kidney (respectively) compared to CPF alone treated animals. Caspase-9 activity in groups co-treated with CPF + IPA1 and CPF + IPA2 was also reduced (p < 0.05) by 66.3% and 110.1% (liver) and by 28.7% and 43.8% (kidney) compared to CPF alone treated rats.
The effect of IPA on the biomarkers of oxidative stress and DNA damage was assessed in the liver and kidney of CPF-treated rats. CPF Administration caused a signi cant decrease in Trx and Trx-R and increased 8-OHdG levels in the liver and kidney of treated rats relative to the control group Figs. 8 and 9.
In the liver, Trx, Trx-R levels in CPF alone treated rats decreased by 27.6% and 90.7%, whereas the 8-OHdG level increased by 58.7% compared to the control group. Contrary, in groups co-treated with CPF + IPA1 and CPF + IPA2, our data show a dose-dependent signi cant increase in Trx (24.3% and 36.5%) and Trx-R (90.3% and 106.6%) levels compared to CPF alone treated rats. There was a signi cant decrease in the 8-OHdG level in the co-treated groups by 13.2% (IPA1) and 79.5% (IPA2) compared to CPF alone group in the liver.
Treatment with CPF signi cantly increased the level of 8-OHdG (68.1%) and decreased Trx (27%) and Trx-R (70.9%) levels in the kidney compared to the control. However, co-treatment with CPF + IPA1 and CPF + IPA2 decreased 8-OHdG by 16.2% and 55.6%, respectively, compared to CPF alone treated rats. In addition, a dose-dependent signi cant increase was observed in the levels of Trx (20.3% and 44%) and Trx-R (89.5% and 124.3%) in CPF + IPA1 and CPF + IPA2, respectively, relative to CPF alone group.

Effects of CPF and IPA on liver and kidney histology of rats
Control: There is no visible lesion. CPF alone: there is centrilobular hepatocellular degeneration and necrosis with Kupffer cell hyperplasia, multifocal necrotizing hepatitis. IPA alone: There is no visible lesion. CPF + IPA1 (25mg/Kg): There is centrilobular hepatocellular degeneration, necrosis and in ammation. CPF + IPA2: There is no observable lesion Fig. 10. Control: there is no noticeable lesion. In the present study, oral administration of CPF resulted in a signi cant reduction in the relative liver weight. However, there was no substantial change in the relative kidney weight in CPF treated rats compared with the control. The decrease in relative liver weight may be an indication of liver toxicity. Our results are consistent with results from previous studies, which also reported a reduction in organ weight (Ezzi et al., 2016;Joshi, Mathur, & Gulati, 2007). Co-administration of IPA improved relative liver and kidney weights in co-treated groups compared to CPF alone treated rats.
The present investigation assessed the levels of serum marker enzymes. These enzymes include AST, ALT, ALP and GGT and are found in the liver under normal physiological conditions. However, cellular stress and tissue injury induced by various toxins could release these enzymes into the bloodstream, indicating liver injury (Kaplan, 1993). In the present study, CPF administration resulted in a statistically signi cant increase in ALP and GGT, indicating hepatic toxicity, resulting in liver necrosis and in ammations (Kuzu et al., 2007). There was, however, a non-signi cant increase in the activities of AST and ALT. Thus, increased activities of hepatic transaminases in serum correlates with hepatocyte injury in CPF treated rats. These ndings are consistent with earlier CPF in experimental rodents (S. A. Mansour & Mossa, 2009;Tanvir et al., 2016). Also, in this study, increases in activities of serum hepatic transaminases observed in CPF treated rats were dose-dependent reduced upon co-treatment with IPA, re ecting the protective potential of IPA against CPF hepatoxicity.
The kidneys play an essential role in the excretion of waste products and toxins such as urea and creatinine. Urea is a by-product of protein degradation, whereas muscles produce creatinine as a byproduct of creatinine breakdown (Dorgalaleh et al., 2013). Both are excreted from the body by the kidney in urine and serve as indicators of renal function. They are also helpful in detecting nephrotoxicity caused by exogenous compounds in living organisms (Sandilands, Dhaun, Dear, & Webb, 2013). This study found a signi cant increase in serum urea level and a non-signi cant increase in serum creatinine level in CPF-treated rats than controls, indicating that CPF caused kidney damage due to various factors, including oxidative stress (S. G. Mansour et al., 2017). The reduced serum urea and creatinine levels in rats co-administered IPA and CPF imply that IPA alleviated CPF-mediated renal dysfunction in the treated rats.
Antioxidants, both endogenous and exogenous, and enzymatic and non-enzymatic, are essential in mitigating oxidative stress. The enzymatic antioxidants (SOD, CAT, GPx, and GST) strongly defend against liver injury and other disorders; thus, they receive a lot of attention (Zhou, Zhang, Yin, Jia, & Shan, 2015). Furthermore, these antioxidant enzymes limit the effects of oxidants in cells and tissues and act in the defence against oxidative cell injury scavenging free radicals (Halliwell & Gutteridge, 2015). Our data indicated a decrease in SOD, CAT, GST and GPx in the liver and kidney of rats treated with CPF alone compared to the control in the current study. The reduction in these antioxidant enzymes may be related to decreases in the synthesis of these enzymes or inactivation/inhibition induced by exposure to CPF (Heikal & H. Mossa, 2012; Owumi & Dim, 2019). This could result in the accumulation of free radicals in the liver and kidney cells of rats treated with CPF alone. Interestingly, our results show an increase in the activities of these antioxidant enzymes in rats co-exposed to CPF and IPA, revealing the antioxidant effects of IPA against CPF mediated oxidative stress in the treated rats.
Reduced glutathione is an endogenous non-enzymatic antioxidant that operates in association with other endogenous antioxidants in preventing oxidative stress. Our results show decreases in the levels of GSH in the liver and kidney of animals treated with CPF alone compared to the control, indicating overutilisation in the detoxi cation process to shield the system from oxidative stress. Consequently, exposure to CPF predisposes the liver and kidney to free radical-mediated cellular damage in rats exposed to CPF. Co-administration with IPA and CPF brought about a signi cant increase in hepatic and renal GSH levels compared to animals treated with CPF alone. IPA has been proven to effectively scavenge hydroxyl radicals effectively and work in tandem with glutathione to minimize oxidative stress (M. Karbownik, J. J. Garcia, et al., 2001). Besides scavenging the hydroxyl radical, IPA has also been shown to scavenge the superoxide anion radical, which aligns with our result as co-administration with IPA and CPF brought about a signi cant increase in hepatorenal GSH levels compared to animals treated with CPF alone. The present study revealed a signi cant decrease in TSH level in the liver and kidney of rats treated with CPF alone compared to the control group, signifying a reduction in antioxidant status and an increased accumulation of free radicals. However, there was a signi cant increase in TSH level in the liver and kidney of rats co-treated with CPF and IPA in a dose-dependent manner compared to the CPF alone treated group, thus showing the antioxidant ability of IPA.
Both ROS and RNS, when present in a typical physiologically system, play essential roles in normal cellular functions such as ghting against infection, regulating different intercellular signalling pathways (Nunes Silva, 2015). However, when present in high concentrations, they overwhelm the innate antioxidant defence system, resulting in oxidative stress, leading to cellular dysfunction via lipid peroxidation, protein, and DNA damage (Martindale & Holbrook, 2002). In this study, rats treated with CPF alone revealed a marked increase in RONS levels compared to the control, implying that CPF activated the formation of free radicals in liver and kidney tissues. A signi cant increase in hepatic and renal LPO levels was also increased in rats treated with CPF alone. The increase in LPO levels can be attributed to the rise in RONS levels due to CPF treatment. Our ndings are in tandem with previous observations

Conclusion
Exposure to CPF can cause hepatorenal toxicity by increased oxidative-in ammatory responses, apoptotic mediators and DNA damage. Co-treatment with IPA reduced hepatorenal toxicities brought about by CPF exposure by decreasing oxido-in ammation stress and apoptosis as depicted in our proposed mechanism of action for CPF toxicity and IPA mitigating roles in Fig. 12. IPA could be used as a potential intervention to minimise hepatorenal toxicities caused by CPF and other organophosphate insecticides acting by these mechanisms.

Declarations Ethical Approval
The experiment was approved by the University of Ibadan Animal Care and Use Research Ethics Committee (ACUREC) UI-ACUREC/033-0521/7 and following the United States National Academy of Sciences guidelines.

Consent to Publish
All the authors listed that participated in the study agreed to the content of the manuscript and the publication thereof.       In uence of CPF alone, IPA alone and their combination on caspase-3 and caspase-9 activities in liver and kidney of rats. CPF: Chlorpyrifos (5 mg/kg); IPA: 3-Indolepropionic acid -IPA1 and IPA2 (25 and 50 mg/kg respectively). Each bar represents the mean ± SD of 6 rats. *: p<0.05 versus control; *: p<0.05 versus CPF alone; ns=not signi cant.

Figure 9
In uence of CPF alone, IPA alone and their combination on the 8-OHdG level in liver and kidney of rats.