Effect of Some Strains of Lactic Acid Bacteria Isolated From Some Fermented Dairy Products on Carbon Tetrachloride-Induced Hepatotoxicity and Nephrotoxicity of Albino Rats.


 The current research was performed to evaluate the potential protective effect of Lactobacillus paracasei ssp paracasei, Pediococcus acidi-lactis and Lactoccus lactis ssp lactis, Sylimarin in the alleviation of health (hepatic and renal) complications caused by carbon tetrachloride (CCl4) in rats. Healthy sixty albino rats were divided into six groups, the first group was control (negative), the second group (control positive) was injected CCl4 (1 ml/kg, 1:1 v/v paraffin oil mixture, i.p. every third day for 8 weeks, The third group (CCl4 + silymarin group) receiving both CCl4 and daily silymarin therapy (50 mg/kg, oral), the fourth group: CCl4+ (Lactobacillus paracasei 1ml orally). The fifth group (CCl4+ Pediococcus acidilactis 1ml orally) and the six group (CCl4+ Lactococcus lactis (1ml orally) for eight weeks per day. Biochemical markers were tested for blood, liver and kidney tissue. Histopathological tests on liver and kidney tissues were performed. The findings obtained have shown that Lactobacillus paracasei ssp paracasei, Pediococcus acidilactis and Lactoccus lactis ssp lactis improved the disrupted biochemical parameters caused by CCl4 therapy. Besides, the findings of the histopathological analysis are in consistent with biochemical parameters and the protective ability of lactic acid bacteria suggesting that the best lactic acid bacteria was Pediococcus acidilactis that helped strengthen liver fibrosis caused by CCl4 therapy, while the best bacterium for improving renal damage was Lactoccus lactis.


Introduction
Lactic acid bacteria (LAB) are commonly known as host health living microorganisms ( (FAO / WHO, 2002). Several in-vitro assays have been successful in the investigation of probiotic potentials of organisms but the use of animal models and human cells have gained much attention recently in the preparations and development of functional foods as the organisms might not replicate such attributes in a living host (Tezel, 2019). lactic acid bacteria are widely used in food fermentation, contributing to the avour, texture and preservation of fermented products. Generally, these bacteria are considered bene cial and some strains may be used for the treatment of human diseases (Elmer et al., 1996). These strains can produce heterologous proteins such as enzymes (lipase and lactase), biological mediators (hormone and interleukin) and molecules that stimulate local immune responses in order to prevent digestive disorders (toxins and viral proteins) can be produced by these strains (Corthier and Renault, 1999).
Lactococcus lactis is a commonly used as a starter strain that, by over expressing the ribo avin biosynthesis genes, can be transformed from a vitamin B2 user to a vitamin B2' factory (LeBlanc et al., 2005). Lactobacillus sp. SBT 2028 had the best antioxidant effects out of 570 strains of LAB (Kaizu et al., 1993) . 19 strains of LAB have antioxidant of 7-12 percent in intracellular cell-free extracts, due to their metal-ionchelating and ROS-scavenging capabilities (Lin and Yen, 1999), Olajugbagbe et al.(2020) indicated that Pediococcus acidilactici isolated from Wara was able to act as a therapeutic agent against a Diarrhoeagenic Enterotoxigenic E. coli and improve gut health . Several scienti c studies have con rmed that the daily ingestion of probiotics or their related products, notably lactic acid bacteria, signi cantly improved human health through a variety of effects, including xenobiotic detoxi cation and competition with pathogenic microbial agents (Maurice et al., 2013), Silymarin is a common herbal extract used as a hepatoprotective agent a mixture of avonolignans obtained from milk thistle (Williamson et al., 1996) , In natural dietary sources, antioxidants are available, and the intake of antioxidants have a number of possible health bene ts (Yeung et al., 2019) .
The liver and the kidney play an important role in the detoxi cation of metabolism and ex-creation of various xenobiotics entering the body,; thus, the toxic effects of chemicals occur mainly in liver and kidney tissues (Abdel-Daim et al., 2013). Chemicals mediated hepatotoxicity that cause liver damage are called hepatotoxins that can rejoin with the basic cellular components that subsequently prompt nearly all forms of liver lesions (Thompson et al . , 2017). Carbon tetrachloride (CCl 4 ) is metabolised in the liver to the trichloromethyl radical (CCl 3 ) by the cytochrome P450 superfamily of monooxygenases (CYP family). This radical subsequently interacts with nucleic acids, proteins and lipids, impairing major cellular processes, leading to altered lipid metabolism (fatty degeneration and steatosis) and reduced amounts of protein (Scholten et al., 2015).
One of the most widely used hepatotoxins in the laboratory study of liver brosis is carbon tetrachloride. The mechanism of liver damage caused by CCl 4 is due to the conversion of CCl 4 into free radical trichloromethyl (CCl 3 *) by activation of drug-metabolising enzymes that trigger lipid peroxidation in the endoplasmic reticulum (Plaa, 2000). The subsequent development of a highly reactive oxygen reaction between CCl3* and cellular molecules and the formation of a trichloromethyl peroxy radical (CCl3OO*) is believed to cause chain reactions that result in CCl4-induced toxicity (Weber et al., 2003;Ilhan and Seckin, 2005). By producing free radicals and increasing lipid peroxidation, carbon tetrachloride has many adverse effects on the liver, kidneys, heart and blood. This is one of the key mechanisms of liver toxicity caused by CCl 4 (Sodergren et al., 2001). As for as we know there are very few studies to date on the protective activity of the liver and kidneys of lactic acid bacteria. Therefore, this study was conducted to determine the protection of the liver and the prevention of lactic acid bacteria against carbon tetrachloride (CCl 4 ) -causing toxicity in rats.

2.1.Materials
Carbon tetrachloride and silymarin were obtained from El-Gomhorya Pharmaceutical Company, Cairo, Egypt and Chemical kits for blood urea nitrogen (BUN), creatinine, protein, albumin, AST, ALT, catalase (CAT) , nitric oxide (NO) and all other chemicals were purchased from Biodiagnostic Company, Cairo, Egypt.

Preparation of LAB strains:
Lactobacillus paracasei, Pediococcus acidilactis and Lactoccocus lactis ssp lactis were isolated from locally produced fermented products (laban zeer, laban rayeb and Karish cheese). These strains were identi ed by some biochemical analyses such as phynotypic characterization of lactic acid bacteria strains( LAB) according to (Bergey manual,1984 andSharpe, 1979) and The API 50CH kit (Biomerieux, Marcy l'Etoile France). They were grown in MRS for rod and M17 for cocci isolated respectively broth for 72 hr at 37 °C to reach their highest growth (Bikheet et. al., 2015). One milliliter of each inoculation was added separate aliquot ( 10 ml ) of skim milk (108 CFU /ml skim milk) for feeding the rats (Zagato et. al., 2014).

2.3.Experimental animals and design
Sixty female (140 ±10 g) Sprague-Dawley albino rats, their age 45 days were obtained from the Animal House of Faculty of Agriculture, Minia University. They were housed under uniform environmental conditions for 10 days until the experiment was started, fed with a daily diet and left to acclimatize to the environment (22 ° C under a light / dark period of 12/12 hours). Animals (each of ten rats) have been divided into six groups and treated as follows: The rst group :Control group (negative), the respective vehicle was injected into rats (0.5 ml / kg b.w saline para n oil).
Then CCl 4 was injected, as de ned in the positive community. Rats were fasted overnight after eight weeks and anesthetized by diethyl ether to take blood samples from all animals in each group from the retro-orbital plexus (Schermer, 1967). Each sample was divided into two parts: the rst part was immediately taken for hematological analysis in a heparinized tube and the second part of the sample was taken in a glass tube and left to coagulate at room temperature for 20 minutes and then centrifuged for 15 minutes at 3000 rpm to obtain serum samples kept at -20 ° C before they were used for evaluation of kidney function and lipid pro le. Animals were then slaughtered and liver and kidney tissues were dissected and washed with ice-cold saline at -20 C, weighed and processed. Liver and kidney tissues in saline were then homogenized and the homogenate was used for the assessment of the oxidative stress marker catalase (CAT) and nitric oxide (NO).
Furthermore, specimens of liver and kidney tissue were xed with 10 percent formalin for histopathological examination.

2.4.Determination of Hematological Parameters
By the Neubauer hemocytometer system (Dacie and Lewis 1991), The numbers of red blood cells (RBC) and white blood cells (WBC) were counted. The concentration of hemoglobin ( Hb) was calculated using the cyanmethemoglobin method in accordance with Jain (1986). The volume of packed cells (PCV) was calculated by Dacie and Lewis (1991) microhaematocrit process.

2.5.Histopathological research
Tissue livers and kidney samples were xed in 10 percent neutral buffered formalin in control animals. They treated classes-cleaning absolute ethyl alcohol for dehydration, trapped in para n, with tap water following serial dilution. Parts 4-5 microns thick were prepared, collected on glass slides for histopathological analysis, depara nized, and stained with hematoxylin and eosin stains also Masson stain (Banchroft et al., 1996).

2.6.Analysis of statistics
The results were expressed as mean ± SD from six parallel measurements and comparisons between treatment means were made utilizing Tukey post hoc test. Variance analysis using ANOVA procedures was performed. GraphPad Prism ® was used (Motulsky, 1999)

3.1.Body weight gain and relative weight of liver and kidneys organs in tested animals
The effect of lactobacillus paracasei (B1), pediococcus acidilactis (B2) and lactococcus lactis (B3) on nal weight , body weight gain, and relative liver and kidney weight of carbon tetrachloride (CCl 4 ) and silymarin treated rats compared to the control group is summarized in Table   1. The body weight gain in CCl 4 and silymarin treated groups decreased signi cantly ( p < 0.05) after eight weeks, while relative weight liver and kidney increased signi cantly compared to the control group, but B1, B2 and B3 treated signi cantly improved growth (Table 1) Data represent the mean ± S.D. of observations from 6 rats. a Signi cantly different from control group at P < 0.05 . b Signi cantly different from CCl 4 group at P < 0.05. c Signi cantly different from CCl 4 + silymarin group at P < 0.05.

3.2.Effect of hematological parameters:
Hematological criteria are used in the routine clinical assessment of the state of patient health to provide valuable information for diagnosis.
The ndings in Table 2 showed that no major differences were found in all groups treated for Hb, RBC, PCV, MCV , MCH and MCHC relative to control levels. While there were signi cant increase in WBC and decrease in Platelets in the group treated with CCl 4 comparing to normal control whereas, animals treated with B2 and B1 plus CCl 4 restored WBC and platelets to nearly normal control (Table2) . Table 3 summarized that the CCl 4 treated group had signi cantly higher serum total protein , globulin ratio, AST, ALT, Urea and creatinine as well as lower albumin compared to other groups. while this elevation was decreased in B1, B2 and B3 groups compared to control group, where B2 group is the best improvement and nearly to control group.

3.4.Oxidative Enzymes:
The data presented in Table 4 showed that the catalase level was signi cantly lower in liver and kidney tissue and that the level of N.O. in liver and kidney tissue increased in the CCl 4 population compared to the control group. However, these therapies (silymarin, B1, B2, B3) improved as liver and kidney catalase levels increased and N.O. Levels relative to the CCl 4 group in liver and kidney tissues (Table 4). The mean ±S.D. is represented by data. Remarks from 6 rats. a Signi cantly different from control group at P < 0.05 . b Signi cantly different from CCl 4 group at P < 0.05. c Signi cantly different from silmarin group at P < 0.05. The mean ±S.D. is represented by data. Remarks from 6 rats. a Signi cantly different from control group at P < 0.05 . b Signi cantly different from CCl 4 group at P < 0.05. c Signi cantly different from siylmarin group at P < 0.05.

4.Histopathological examination
Histological studies of the hepatoprotective effects of L.paracasei , P. acidilactaci and L. lactis caused liver damage to CCl 4 are shown in (Figure 1, 2). Hepatocytes (H) and central vein (CV) are divided into anastomosing cords or plates, separated by anastomosing hepatic sinusoids (S). Sections of the liver of the control rat have been revealed. CCl 4 injection induced many changes in the histological liver, such as disruption of the general morphology of the hepatic lobule. The central veins surround numerous areas of collagen ber deposition between hepatocytes (arrows). Improvement in liver histology has been shown in the administration of L.paracasei , P. acidilactaci and L. lactis plus CCl 4 , Most regions seem to have recovered, well-preserved hepatocytes, and no are together wia of necrosis.

Discussion
Recent scienti c studies have shown that probiotics protect against sequences of various liver diseases such as cirrhosis / cirrhosis, hepatic encephalitis, and alcoholic and non-alcoholic fatty liver disease. In the present study, 3 lactic acid bacteria were selected with a strong antioxidant effect . this study explained that a hepatic and renal protective effect of the L. paracasei, P. acidilactaci and L. lactis in rats induced by CCl 4 . Therefore, hematological parameters, liver and kidney function and histopathological tests were performed on liver and kidney tissue. The results obtained have shown that the disturbed biochemical parameters caused by CCl 4 therapy boost three lactic acid bacteria. In addition, the results of the histopathological study are consistent with the biochemical parameters and protective capacity of lactic acid bacteria, indicating that the best P. acidilactaci (B2) was a lactic acid bacterium that helped improve CCl 4 therapy-induced liver brosis, while the best of these was L. lactis (B3) against renal damage to CCl 4 . Body weight changes may provide an indicator of drug effect and are used for assessment of responses to the drug therapy (Asuquo et al., 2012),the rise in kidney and liver weight in group treated CCl 4 may be attributed to xenobiotics-related lesions and injuries, which per oxidize proteins (Wong et al., 2010). Also the increase in WBC can be due to the stimulation of the immune defense system (Kashinath 1990) or increasing antigen concentration in the body (Hoeney, 1985), the low values PCV may be attributed to anemic conditions. decreasing hemoglobin in injected rats with CCl 4 is an indication of hemolysis and the decreasing in hemoglobin has a corresponding elevation in met hemoglobin content which affects the oxygen-carrying the blood, caused by the toxicant (Tilak et al, 2007). On the other hand Mandour et al.(2020) showed that the time effect had signi cant differences in the values of WBCs and found the WBC , Hb concentration and PLT count increased in goats treated with probiotic at T3 and T6 compared to its values at T0 These results are in the same line with the previous research by Salatin et al. (2019). Increased total protein synthesis, which is characteristic of advanced age and may be due to liver compensation for more pronounced proteinuria, elevated proteolysis or accumulation of altered proteins ( Van et al . , 1977). Oumeddour et al ( 2019) reported that rats were prevented from CCl 4 -induced hyperglycemia by the administration of probiotic bacteria. Even though the existence of bioactive components has been hypothesized to inhibit beta-cell apoptosis and increase insulin secretion (Al-Hariri, 2011). Treatment with the probiotics did not affect any of the parameters measured when compared with the control group, indicating that the probiotics did not affect liver integrity (Jantararussameeetal.,2020).
Results obtained in this study are in the same line with Salatin et al. (2019), and Oumeddour et al. (2019) they are also in accordance with Fahmy et al.(2016) and Al-Sayed et al . ( 2015) stated that CCl 4 increases biochemical parameters such as AST, ALT, urea and creatinine serum levels. The high level of liver enzymes in albino rats injected with CCl 4 may be due to alkylating DNA structures disrupting the hepatocyte membrane architecture, causing DNA damage and subsequent cell degeneration, resulting in leakage from damaged liver tissues, overproduction, and leakage in circulation, and decreased hepatic clearance (Abdo et al., 2015); (Mohamed et al., 2016); The increasing levels of creatinine and urea may be due to a diminish in glomerular ltration rate caused by acute renal dysfunction (Rahmat et al., 2014).
Biological antioxidants are compounds that protect biological systems against the potentially harmful effects of processes or reactions which can cause excessive oxidation (Krinsky 1992) It is also possible to refer to them as scavengers (Salvemini and Botting1993). Catalase (CAT) is a hem protein that catalyzes the reduction of hydrogen peroxide and protects tissues from highly reactive hydroxyl radicals (Searle and Wilson, 1980).The decrease in the activity of CAT can result from the activation of the enzyme by glycation. (Yan and Harding, 1997).
Results obtained in this study are in good agreement with what obtained by Oumeddour et al . ( 2019) who showed that treatment with CCl 4 resulted in a major de ciency in the liver catalase of the enzymatic antioxidant defense mechanism, Also Heeba and Mahmoud (2014) showed that no signi cantly increase when rats treated with CCl4. lactic acid bacteria having an anti brotic effect, the anti brotic function of silymarin has been con rmed by several studies, In line with the present ndings, the anti brotic effect of silymarin on liver brosis induced by dimethyl nitrosamine showed a decreased level of collagen after treatment with silymarin (Joseph et al . 2006) and a major inhibition and reversal of the progression of hepatic brosis induced by dimethyl nitrosamine (Zhao et al., 2006). Youssef et al.(2010) showed that necrosis, hepatocyte vacuolization, ballooning degeneration with dilation and congestion of branches of the hepatic portal vein and sinusoids of the liver were caused by CCl 4 . In the portal regions, increased in ammatory cellular in ltration was observed and was time-dependent. Many authors have reported similar ndings and shown that CCl 4 caused in ammatory cell invasion, patchy areas of necrosis, and rat liver brotic and cirrhotic changes (Zerin et al., 2004;Abdel Salam et al., 2007). The hepato-protective function of silymarin in various toxic models of experimental liver diseases is that of antioxidant, ant lipid per oxidative, anti-brotic, antiin ammatory, membrane stabilizing, immune modulatory and liver regenerating pathways (Chlopcikova et al., 2004;Pradhan and Griish, 2006). Jantararussamee et al. (2020) found that rats receiving probiotics or silymarin had signi cantly lower serum enzyme levels, less in ammation and less brosis. Liver damage was lower in the thioacetamide plus probiotics treated population. Consumption of a mixture of probiotic lactic acid bacteria (a mixture of Lactobacillus paracasei,Lactobacillus casei and Weissella confusa) reduces liver brosis production.These ndings agree with Naima et al. (2014) who reported that CCL 4 induced nephrotoxicity of 0.5 ml / kg (twice a week for six weeks) and 5 ml / kg (once a week for six weeks) of CCL 4 respectively in rats during administration.Other research performed by Eric and Adolphus (2020) showed that, relative to the control group, no signi cant histopathological differences were found in the kidney histology of all the animal groups.This may be due to the length of administration (twice a week for one week) of 0.5 ml / kg CCL 4 .

Conclusion
In conclusion, the results of this study ndings strongly support administration of lactic acid bacterialactococcus lactis (B3) and pediococcus acidilactis (B2) than Lactoba-cillus paracasei (B1), can signi cantly protect the liver and kidneys from CCl 4 -induced toxicity by increasing the pathway of ant oxidative' stress.