Effect of thiram on rat kidney: inhibition of brush border membrane and antioxidant enzymes, diminution of antioxidant capacity, enhanced DNA damage and DNA-protein cross-linking

Thiram is a dithiocarbamate pesticide that is widely used as a fungicide to protect crops and seeds, especially in China and India. Although thiram is considered relatively safe for humans but due to its persistent nature it may become a health hazard for human beings and animal if long term exposure takes place. The aim of the present work was to study the effects of oral administration of thiram on kidney of male rats given different doses of thiram (100, 250, 500, 750 mg/kg body weight) for 4 consecutive days. This treatment signicantly reduced cellular glutathione and total sulfhydryl content but enhanced protein carbonyl and hydrogen peroxide levels. The plasma creatinine and BUN levels were also elevated indicating nephrotoxicity. The activities of antioxidant enzymes catalase, superoxide dismutase, glutathione peroxidase, thioredoxin reductase and glutathione-S-transferase were signicantly decreased. The antioxidant capacity was diminished resulting in less free radical quenching and metal reducing ability of kidney. Administration of thiram also led to inhibition of intestinal brush border membrane enzymes: alkaline phosphatase, leucine aminopeptidase, γ-glutamyl transferase and maltase. Activities of enzymes of glucose metabolism viz. glycolysis, citric acid cycle, pentose phosphate pathway and gluconeogenesis were also inhibited. Histopathology of kidney tissue revealed tubular dilation, tubular cast, breaking of apical cytoplasm and intestinal hemorrhage. A signicant increase in DNA fragmentation, DNA strand breaks and DNA-protein cross-linking was also observed in thiram treated rats compare to control group. These changes in kidney could be due to marked perturbation in antioxidant defense system induced by free radicals generated upon exposure to thiram.


Introduction
Dithiocarbamate fungicides form a large group of sulfur containing chemicals that have numerous uses in agriculture and medicine (Buac et al., 2012;Thind and Hollomon, 2017). They can be applied to the foliage of plants for seed and fruits treatment and also used as bird and rodent repellents.
Tetramethylthiuram disul de, commonly known as thiram, is a dithiocarbamate fungicide that exhibits a broad spectrum of antifungal activity. It is widely used in agriculture due to its low cost, good e cacy and relatively low toxicity. It is also used as slimicide in water cooling systems, in sugar, pulp, and paper manufacturing and as vulcanization accelerator in the rubber industry (Alam et al., 2017;Cereser et al., 2001a). Thiram is also employed in the treatment of human scabies, as a sun screen and as a bactericide applied directly to the skin or incorporated into soap. Thiram has metal chelating properties because of which it is used as a scavenger in waste water treatment (Kanecoa et al., 2009). In agricultural eld it is used to control colletotrichum lint on ax, fungal diseases on sa ower and cotton seed, as a repellent for rabbit, deer and black birds. Thiram is applied on seeds to control black root of sugar beet, apple scab, grey mould of strawberries, brown rot of stone fruit, against damping off of nursery seedlings and turf crops (Sharma et al., 2003).
Despite several useful applications, the excessive use of thiram is harmful as it can enter into the environment by surface run-off, spraying on crops or in gardens, also via e uents of waste water treatment plants and can get incorporated into the food chain. People can be exposed to thiram by consuming food and liquid containing residues, by inhaling thiram contaminated air or by occupational exposure. Thiram exposure is associated with several toxicological effects including endocrine disruption (Chen et al., 2018), hepatotoxicity (Saqib et al., 2005) neurotoxicity (Agrawal et al., 1997) and genotoxicity (Perocco et al., 1989). Thiram also interferes with the metabolism of xenobiotic compounds by inhibiting the enzyme arylamine N-acetyltransferase-1 (Xu et al., 2017). Thiram and ziram induce neurotoxicity by increasing the intracellular level of Ca 2+ through non-selective cation channels (Han et al., 2003).
Ingestion of seeds coated with thiram and imidacloprid decreases cellular immune response in redlagged partridges (Alectoris rufa) (Lopez-Antia et al., 2015). Stoker et al. (1993) showed that thiram affects oocyte fertilization by delaying ovulation. About one million birds suffer from depressed growth, soft egg shell and leg abnormalities from consuming thiram contaminated feed (Rath et al., 2005;Nageswara et al., 2017). At 100-500 ppm, thiram also inhibits the laying of eggs in hens, partridges and quails (Lorgue et al., 1996). Thiram is also a well know inducer of tibial dyschondroplasia, a common metabolic cartilage disease found in quickly growing poultry birds (Waqas et al., 2020). Since thiram can enter surface water by runoff and erosion its effects on the aquatic ecosystem have also been studied.
Exposure to thiram at the early life stage affects the development of zebra sh (Danio rerio) (Chen et al., 2018). It decreases growth rate and induces neurotoxicity in Daphnia magna, a small planktonic crustacean (Belaid et al., 2019).
A strong correlation between pesticide toxicity and enhanced production of reactive oxygen species (ROS) and free radicals has been observed (Khan et al., 2005;Mansour and Mossa, 2010). Oxidative stress is characterized by an increase in the levels of ROS and free radicals that exceed the capacity of cells to quench them. Thiram also induces oxidative stress by generating ROS and free radicals (Grosicka-Maciag et al., 2008;Cereser et al., 2001b;Kurpios-Piec et al., 2015). The formation of these reactive species is the main cause for the pathogenesis of many diseases such as cancer, neurodegeneration, cardiovascular diseases and rheumatoid arthritis (Valko et al., 2007).
Due to the functional, biochemical and morphological heterogeneity of the kidney, several drugs and xenobiotics show site speci c toxicity to this organ (Werner et al., 1995). Indeed, pesticides can cause severe renal damage including in ammation, tubular cell toxicity, crystal nephropathy (Zager, 1997;Schetz et al., 2005) and nephrotoxicity in rat model (Behling et al., 2006). Blood chemistry showed that thiram causes kidney damage in rats and beagle dogs (Maita et al., 1991;Lee et al., 1978). However, except for these two studies there are no reports investigating the nephrotoxic effect of thiram. In view of the paucity of reports on toxicity of thiram on kidney, we have examined the biochemical, histological and genotoxic potential of this fungicide on rat kidney.

Material And Methods
Chemicals Thiram (purity, 97%) was purchased from Sigma Aldrich, USA. All other chemicals used were of analytical grade and obtained either from Sisco Research Laboratories (Mumbai, India), Himedia Laboratories (Mumbai, India) or Sigma-Aldrich (USA).

Experimental protocol
The animal experiments were done as per the guidelines provided by the Institutional Ethics Committee (IEC) of Aligarh Muslim University that monitors research involving animals (R. No. : 714/GO/Re/S/02/CPCSEA). Adult male Sprague Dawley rats weighing 150-200 g were obtained from National Institute of Biologicals, Noida, India. The animals had unlimited access to standard pellet rat diet and clean drinking water. After one week of acclimatization, 30 male rats were randomly divided into ve groups of six animals each. Thiram was dissolved in corn oil and orally administered (by gavage) for 4 days at doses of 100, 250, 500, 750 mg/kg body weight/day at an interval of 24 h. Untreated animals in the control group received an equivalent volume of corn oil by gavage. There was no animal mortality because these doses were below the LD 50 value of thiram for rats (620 to over 1900 mg/kg body weight).
All animals were sacri ced 24 h after the administration of last dose of thiram, under anesthesia. Both kidneys were carefully removed from each animal, decapsulated and used for the preparation of homogenates. Blood was removed from the heart using a syringe and transferred to heparinized tubes.

Renal function test
Plasma concentrations of creatinine and blood urea nitrogen (BUN) were used as markers of renal function. Blood was centrifuged at 1000 rpm for 10 min at 4ºC and supernatant (plasma) was saved. The plasma was rst deproteinized by adding 3% trichloroacetic acid in 1:3 ratio and centrifuged at 3000 rpm for 5 min to pellet the precipitated proteins. The supernatant was used for BUN and creatinine estimation using kits purchased from Arkray Healthcare, Mumbai, India. Creatinine was determined by modi ed Jaffe's method (Haugen, 1953) and BUN by diacetyl monoxime reagent (Rosenthal, 1955).

Preparation of kidney homogenates
Each kidney was divided into cortex and medulla using a sharp scalpel. The cortex and medullary portions were homogenized in 2 mM Tris, 50 mM mannitol buffer, pH 7.5, to prepare a 10% (w/v) homogenate. Homogenates were further homogenized using an Ultra Turrex, Kunkel homogenizer by passing ve pulses of 30 sec each. Each homogenate was divided into aliquots and used immediately or quickly frozen at -80°C for further analyses.

Preparation of brush border membrane and assay of bound enzymes
The cortical homogenates prepared above were further processed to isolate brush border membrane (BBM) vesicles. To each homogenate 10 mM MgCl 2 ( nal concentration) was added, samples left for 20 min on ice with continuous stirring and then centrifuged at 2000 rpm for 15 min at 4ºC. The supernatant so obtained was centrifuged at 17,000 rpm (35000 x g) at 4°C for 30 min. The supernatant was discarded and pellet was suspended in 300 mM mannitol, 5 mM Tris-HCl buffer, pH 7.5, homogenized in a hand held homogenizer and again centrifuged at 17000 rpm for 30 min at 4°C. The supernatant was discarded and the white uffy layer present in pellet, containing BBM, was suspended in 300 mM mannitol, 5 mM Tris-HCl, pH 7.5 buffer (Khundmiri et al., 1997). The BBM preparations were used immediately or stored at -80ºC and used within 3-4 days. Protein concentration in homogenates and isolated BBM vesicles was determined using Folin's phenol reagent (Lowry et al., 1951).

Non-enzymatic oxidative stress markers
The concentration of reduced glutathione (GSH) was determined in protein-free homogenates using 5,5'dithiobis-2-nitrobenzoic acid (DTNB), as described by Beutler et al. (1963). The total sulfhydryl group content was quanti ed from the concentration of the yellow thionitrobenzoate anion, which forms upon reaction of sulfhydryl/thiol groups with DTNB and absorbs maximally at 412 nm (Sedlak and Lindsay, 1968). Protein oxidation was determined from carbonyl content as described by Levine et al. (1990). In this method, protein carbonyl groups react with 2,4 dinitrophenylhydrazine to form hydrazone adduct which was quanti ed from absorbance of solutions at 360 nm. Hydrogen peroxide (H 2 O 2 ) concentration was determined by the method of Gay and Gebicki (2000). H 2 O 2 oxidizes Fe 2+ to Fe 3+ under acidic conditions; Fe 3+ reacts with xylenol orange to form a purple complex that absorbs at 560 nm. All parameters were determined in kidney homogenates.

Antioxidant capacity
The antioxidant power (AO)/capacity of cortex and medullary homogenates was determined by using FRAP (ferric reducing antioxidant power) and DPPH (2,2-diphenyl-1picrylhydrazyl) assays. In FRAP assay, 0.1 ml homogenate was added to 1.5 ml FRAP reagent (300 mM sodium acetate, 10 mM 2,4,6-Tris(2pyridyl)-s-triazine, 20 mM FeCl 3 , pH 3.6). The absorbance of samples was recorded after 5 min at 593 nm (Benzie and Strain, 1996). In DPPH assay, the AO power of homogenates was determined in terms of percent quenching of DPP • radical. Lower the quenching of DPP radical, lesser is the AO power. To 0.1 ml homogenate were added 0.4 ml of 10 mM sodium phosphate buffer, pH 7.4, and 0.5 ml of 0.1 mM DPPH reagent. The samples were kept for half an hour in the dark, centrifuged at 12,000 x g and absorbance of supernatants was read at 517 nm (Mishra et al., 2012).

Antioxidant enzymes
The activity of catalase (CAT) was determined from its ability to convert H 2 O 2 into H 2 O (Aebi, 1984) and Cu, Zn-superoxide dismutase (SOD) from the inhibition of autoxidation of pyrogallol (Marklund and Marklund, 1974). Glutathione reductase (GR) was assayed from the reduction of oxidized glutathione (GSSG) to GSH with concomitant conversion of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to its oxidized form (NADP + ) as described by Carlberg and Mannervik (1985). Thioredoxin reductase (TR) was assayed from the reduction of DTNB to yellow thionitrobenzoate anion in presence of NADPH; the absorbance of yellow color produced was read at 410 nm (Tamura and Stadtman, 1996).
Glutathione peroxidase (GPx) was assayed by the method of Flohe and Gunzler (1984). In this method NADPH is oxidized to NADP + , in presence of GSSG and GR, resulting in decrease in absorbance at 340 nm. Glutathione-S-transferase (GST) is a phase II detoxifying enzyme which acts by conjugating harmful xenobiotics to GSH. It was assayed using 1-chloro-2,4-dinitrobenzene and GSH as substrates. The absorbance of the conjugate formed was read at 340 nm. An extinction coe cient of 9,600 M -1 cm -1 was used to calculate enzyme activity (Habig et al., 1974).

Metabolic enzymes
Hexokinase activity was determined from the decrease in concentration of free glucose as described by Crane and Sols (1953). Lactate dehydrogenase (LDH) was assayed from the oxidation of reduced nicotinamide dinucleotide (NADH) to its oxidized form (NAD + ) in the presence of sodium pyruvate; the decrease in absorbance of solution was read at 340 nm (Khundmiri et al., 2004). Glucose 6-phosphate dehydrogenase (G6PDH) was assayed from the conversion of glucose 6-phosphate to 6phosphogluconolactone, with concomitant reduction of NADP + to NADPH (Shonk and Boxer, 1964). The activity of gluconeogenic enzymes, glucose 6 phosphatase (G6Pase) and fructose 1,6-bisphosphatase (FBPase), were determined from the release of inorganic phosphate from their substrates, glucose 6phosphate and fructose 1,6-bisphosphate, respectively (Shull et al., 1956;Freedland and Harper, 1959). The amount of inorganic phosphate released was estimated by using Taussky and Shorr reagent (Taussky and Shorr, 1953). Malic enzyme was assayed from the oxidative decarboxylation of L-malate to pyruvate. NADP + serves as electron acceptor and is converted to NADPH resulting in increase in absorbance of solution at 340 nm (Ochoa et al., 1948). Malate dehydrogenase was assayed by following the oxidation of NADH to NAD + using oxaloacetate as substrate. The decrease in absorbance of solution was recorded at 340 nm (Mehler and Korenberg, 1948).

Acid phosphatase and total ATPase
Total ATPase activity was determined from the concentration of inorganic phosphate released upon hydrolysis of ATP (Bonting et al., 1961). Acid phosphatase acts on p-nitrophenyl phosphate to from yellow p-nitrophenol that absorbs at 415 nm (Mohrenweiser and Novotny, 1982).
DNA fragmentation (DF) and DNA-protein crosslinks (DPCs) DNA damage by thiram was assayed by diphenylamine and DNA-protein cross-linking (DPC) assays. The concentration of fragmented DNA was estimated using diphenylamine as described by Burton (1956). The homogenates were centrifuged and 0.5 M perchloric acid was added to both supernatant and pellet to precipitate the proteins. The samples were centrifuged and to 0.5 ml supernatant, 1 ml diphenylamine reagent (1.5 gm diphenylamine, 100 ml acetic acid, 1.5 ml H 2 SO 4 and 1.6 µg/ml acetaldehyde) was added. After 16-20 h at room temperature in the dark, absorbance of solutions was read at 600 nm.
DPCs are formed when cells are exposed to DNA damaging agents. The levels of DPCs were determined using K + /SDS assay, exactly as described by Zhitkovich and Costa (1992).

Comet assay
The genotoxic effect of thiram was analyzed by single cell gel electrophoresis using the method of Singh et al. (1988) but with slight modi cations. Single cell suspension was prepared by mincing the tissue in Rosewell Park Memorial Institute 1640 medium. A fraction of suspension was mixed with 1% low melting point agarose (LMPA), poured over glass slides coated with 1% normal agarose and allowed to solidify.
Another layer of LMPA (0.5%) was then spread over slides which were then dipped in chilled lysis buffer (100 mM EDTA, 2.5 m NaCl, 10 mM Tris-HCl, 1% Triton-X-100, pH 10.0) for 3 h in dark at 4ºC. The slides were then transferred to electrophoresis buffer (1 mM EDTA, 300 mM NaOH, pH 13) for 30 min to allow DNA unwinding. Electrophoresis was carried out for 30 min at 0.7 V/cm and 300 mA at 4ºC. The slides were then neutralized with 0.4 M Tris-HCl buffer, pH 7.4. The DNA was stained by adding a drop of 20 µg/ml ethidium bromide on slides which were then washed with distilled water. The DNA was visualized under uorescence microscope at 100 x and analyzed with Comet assay software from CaspLab.

Histopathology
Longitudinal sections of the kidney of 10 µm thickness were cut. The tissue was processed to prepare slides as described by Culling (2013) and stained with either eosin or hematoxylin. The slides were then visualized under microscope at 400 x magni cation.

Statistical analysis
All data was expressed as mean ± standard error mean for six rats in each group and statistical signi cance was reported using one way ANOVA. Student t-test was used for comparison between treated and control groups. Differences were considered signi cant at p < 0.05.

Renal function test
Both BUN and creatinine levels showed a thiram dose-dependent increase. The concentration of BUN in plasma increased to 1.6 fold in treated group (Fig. 1A) while creatinine increased by 1.32 fold, compared to untreated rats (Fig. 1B).

BBM enzymes
The effect of thiram on the speci c activities of BBM marker enzymes in isolated BBMV and cortical and medullary homogenates is shown in Tables 1 and 2. Thiram exposure caused a signi cant reduction in the speci c activities of all BBM enzymes in cortical and medullary homogenates. At the highest dose of thiram (750 mg/kg body weight) the enzyme activities, relative to control, were: ALP, 44.50 and 43.59%; LAP, 46.73 and 45.02%; GGT, 58.38 and 55.15%; maltase, 39.48 and 45.83%. A similar pattern was also evident in the activities of these four BBM enzymes in isolated vesicles: ALP, 40.29%, LAP, 43.63%, GGT, 51.47% and maltase, 31.31%.

Non-enzymatic oxidative stress markers
The levels of both GSH ( Fig. 2A) and total SH (Fig. 2B) showed a thiram dose-dependent decrease. While a signi cant increase in carbonyl content (Fig. 2C) was seen along with an increase in H 2 O 2 level (Fig.   2D) in rats of thiram treated groups, compared to control group. The percent levels, relative to control, in these parameters at the highest dose of thiram (750 mg/kg body weight), in cortical and medullary homogenates, were: GSH, 26.2 and 20.5; total SH, 50.0 and 55.6; protein carbonyls, 261.4 and 277.3; H 2 O 2, 160.8 and 170.4.

Antioxidant capacity
AO capacity of cortex and medulla decreased to 50.70% and 54.12%, respectively, as determined by the FRAP assay (Fig. 3A). In DPPH method, a reduction in DPPH quenching ability of cortical (46.25%) and medullary (42.05%) homogenates was observed with increase concentration of thiram (Fig. 3B).
Antioxidant enzymes SOD catalyzes the dismutation of superoxide anion to H 2 O 2 , which is then converted to oxygen and water by CAT and GP. The speci c activities of all three enzymes show a dose-dependent decline in cortical and medullary homogenates of thiram treated rats. The SOD, CAT and GP activities, as percent control, were: 34.78 and 23.13, 48.11 and 46.71, 56.89 and 58.97, respectively. The speci c activities of secondary AO enzymes (GR, TR and GST) also decreased in a similar thiram dose-dependent manner. The percent activities of these enzymes, relative to control, were: GR, 48.20 and 32.79; TR, 36.24 and 43.67 and GST, 42.24 and 40.41 (Table 3).

Metabolic enzymes
The speci c activities of hexokinase and G6PDH showed a thiram dose-dependent decrease in both cortex and medullary homogenates: Hexokinase (41.58% in cortex and 48.54% in medulla) and G6PDH (46.66% in cortex and 62.06% in medulla). The activity of LDH increased and was 1.58 fold in cortical and 1.73 fold in medullary homogenates, relative to control. Similarly, the activities of gluconeogenic enzymes FBPase and G6Pase showed a thiram dose-dependent decrease. The activity of FBPase decreased to 33.33% in cortex and 39.43% in medulla while G6Pase decreased to 42.37% in cortex and 46.15% in medullary homogenates. The activity of malate dehydrogenase, also showed a gradual decrease. The percent decrease in its activity was 37.12 in cortex and 34.28 in medullary homogenate. Like LDH, the activity of malic enzyme also increased with increase concentration of thiram. A 1.37 fold increase in the activity of malic enzyme was observed in cortex and a 1.45 fold increase in medullary homogenate (Table 4).

Acid phosphatase and total ATPase
Both acid phosphatase and total ATPase showed a thiram dose-dependent decrease in activity. The activity of ACP declined to 53.40% in cortex and 59.65% in medullary homogenates (Fig 4A). Total ATPase was 40.20% in cortex and 33.74% in medullary homogenates, relative to control values (Fig 4B).

DNA damage
The genotoxic effect of thiram was determined by diphenylamine, DPC and comet assays. The levels of fragmented DNA increase and was 9.7 fold in cortex and 10.8 fold in medullary homogenates, as measured by diphenylamine assay (Fig. 5). Similarly the levels of DPCs also increase in thiram treated group compare to control group. The DPC increased by 2 fold in cortical and >2 fold in medullary homogenates (Table 5). An increase in tail length of DNA in comet assay was seen in a thiram dosedependent manner. A 12.6 fold increase in comet tail length of DNA, relative to control, was seen at the highest dose of thiram given to rats (Fig. 6).

Histopathology
Histopathology of kidney tissues reveals normal structure in control group but a progressive damage in tubular epithelia was observed at all doses of thiram. At 500 and 750 mg thiram/kg body weight, cellular debris in tubular lumen, formation of tubular cast, saw tooth appearance and interstitial hemorrhage were evident in cortex region of kidney (Fig 7).

Discussion
BUN and creatinine are metabolic waste products which are routinely excreted out of the body by kidney. A moderate increase in plasma creatinine and BUN levels was recorded in thiram treated rats. This could be due to decrease in glomerular ltration that may have occurred upon reduced reabsorption by renal tubules because of degeneration of tubular epithelial cells. A similar trend in plasma BUN level was observed on oral administration of thiram in rats in a study conducted by Lee et al. (1978).
A signi cant reduction in the activities of BBM enzymes; ALP, LAP, GGT and maltase was observed with increase in dose of thiram in isolated BBMV and cortical homogenate. The decline in the speci c activities of these enzymes was more in isolated BBMV than in the homogenate. ALP and LAP are metalloproteins whereas maltase and GGT have cysteine residues that are involved in binding of substrate at the enzyme active site. The decrease in the activities of BBM marker enzymes could either be due to metal chelating property of thiram or by formation of mixed disul de with the critical cysteine residues, or their oxidation, present at the active site of enzyme (Marikovsky, 2002;Xu et al., 2017). Another reason could be loss of the enzymes into the lumen of the tubule upon membrane damage. GSH, a tripeptide (glutamyl-cysteine-glycine) non-enzymatic antioxidant protects cells from free radicals. Within the cells GSH is found both in free form and bound to proteins. A gradual decrease in the concentration of GSH and total SH was observed with increase in dose of thiram. This may be due to oxidation of thiol group of GSH by ROS generated by thiram, conversion to GSSG or formation of disul de bridge with the thiram molecule. Another reason could be the reduced activity of enzyme GR that regenerates GSH from GSSG (Cereser et al., 2001b). The increase in the levels of H 2 O 2 could be due to decrease in the activities of AO enzymes CAT and GP that under normal conditions convert H 2 O 2 into H 2 O and O 2 . Carbonyl groups formed upon oxidation of side chain of amino acids are markers of protein oxidation. A thiram dose-dependent increase in the protein oxidation was observed which could be due to increase concentration of H 2 O 2 that will lead to the formation of highly reactive hydroxyl radical (Parvez and Raisuddin, 2005).
Thiram exposure caused a signi cant inhibition in the activities of primary (SOD, CAT and GP) as well as secondary (GR, TR and GST) AO enzymes. Thiram can chelate copper ion so inhibition in the enzymatic activity of SOD could be due to sequestering of copper ion by thiram (Babo and Vasseur, 1992). CAT and GP convert H 2 O 2 into H 2 O and O 2 and the latter does so by conjugating it with GSH. The decrease in the activity of enzyme GP could either be due to attack of ROS on selenium atom or carbomylation of cysteine residue present at the active site. CAT can be inactivated by singlet oxygen generated from excess H 2 O 2 (Kim et al., 2001). Elskens and Penninckx (1995) have reported that, in the presence of NADPH, thiram acts as an inhibitor of GR. TR/thioredoxin system maintains thiol/disul de redox balance in the cell and protects them from oxidative damage. A highly conserved sequence (Cys-Gly-Pro-Cys) is present at the active site and modi cation of cysteine residues by thiram may be a reason for decreased activity of TR. GST is a major phase II detoxi cation enzyme. It detoxi es harmful xenobiotics that enter the human body by conjugating them with GSH. Thiram, like other sufhydryl reagents (Smith and Litwack, 1989) may reduce the binding a nity of substrate, 1-chloro 2,4-dinitrobenzene without altering the binding of glutathione and thus, decrease the activity of GST.
A dose-dependent decline in the non-enzymatic AO power of kidney cells also took place on exposure to thiram. Quenching of DPP • radical (DPPH assay) and reduction of ferric ion into ferrous ion (FRAP) by AOs present in cortex and medullary homogenate was used as a measure to quantify the AO power. A signi cant reduction in AO power was observed in both these assays. The reason may be diminished enzymatic (AO enzymes) and non-enzymatic (GSH) AO defense system of the cell in thiram treated animals.
Hexokinase catalyzes the rst step of glycolysis; it converts glucose to glucose 6-phosphate and commits glucose to catabolism. The decreased activity of hexokinase, could be due to the interaction of thiram with the free SH group present at the active site of this enzyme and essential for its catalytic function (Stromme, 1963). LDH converts pyruvate to lactate and regenerates NAD + . Increased activity of LDH could be attributed to its dissociation from membrane and its release into the cytosol (Rana and Shivanaddappa, 2010). Devi et al. (2020) showed that binding of pesticides (rotenone and chlorpyrifos) induces alteration in the secondary structure of malate dehydrogenase and these conformational changes lead to the formation of cytotoxic conformers that generated oxidative stress. So, decrease in the activity of malate dehydrogenase could be due to alteration in its structure occur due to binding of thiram. Superoxide radical generated by reaction of H 2 O 2 with Fe 2+ can inhibit the enzymatic activity of G6PDH (Bagnasco et al., 1991). Another reason could be decreased activity of hexokinase. Malic enzyme decarboxylates malate to pyruvate with concomitant reduction of NADP + to NADPH. NADPH has two important functions: it maintains the redox environment of cell and is involved in the synthesis of fatty acids. The increase in the activity of malic enzyme may be a defense mechanism of cell to protect itself from ROS by generating more NADPH (Corpas and Barroso, 2014). A signi cant reduction in the activities of gluconeogenic enzymes, FBPase and G6Pase was seen with increase in dose of thiram. FBPase convert fructose 1,6-bisphosphate into fructose 6-phosphate while G6Pase dephosphorylate glucose 6phosphate to glucose. The decrease in activity of G6Pase could be due to sequestering of metal ion by thiram (Nordlie and Johns, 1968) and that of FBPase due to increased generation of H 2 O 2 (Halliwell and Gutteridge, 1985).
ATPases are membrane bound enzymes that hydrolyze ATP to ADP and utilize the energy generated during this reaction to transport cations across the cell membrane. The decrease in the activity of ATPases may be due to the ability of dithiocarbamate to form complexes with metal activated enzymes (ATPases and oxidoreductase) (Owens, 1969) or changes in lipid composition and uidity of plasma membrane (Pentyalat and Chatty, 1993). Acid phosphatase performs diverse biological functions including microbial killing, bone resorption, phosphate acquisition in plants and iron transport in animals. The activity of ACP widely depends on the reduced state of thiol group of cysteine residue present at its active site. Oxidation of this critical cysteine residue by ROS such as H 2 O 2 may reduce the catalytic activity of the enzyme (Gazo et al., 2015).
The genotoxic effect of thiram was studied using several methods. The level of fragmented DNA increased in a thiram dose-dependent manner and these result are consistent with a previous study conducted by Rath et al. (2005) in which they showed that thiram exposure induces DNA damage in tibial growth plate. A similar trend was observed in the other two assays used to study the genotoxic potential of thiram. DPCs are formed when cells are exposed to xenobiotics like pesticides and are known to inhibit DNA metabolic processes such as replication and transcription. An error in DNA metabolic processes results in the formation of sister chromatid exchange (SCE). Pienkowska and Zielenska (1990) showed that thiram increases the frequency of SCE in human lymphocytes from this we can infer that thiram may led to the formation of DPC. The comet assay is a sensitive method for detecting low levels of single strand breaks. A signi cant increase in tail length of DNA was observed in a thiram dose-dependent manner that corroborates the gentoxic effect of thiram (Emmanouil et al., 2008). Since previous studies have shown the pro-oxidant nature of dithiocarbamate compound so we hypothesize that the thiraminduced DNA damage could be due to a compromised redox environment and greater generation of ROS that directly modify DNA bases and the deoxyribose sugar. The other reason could be the alkylation of DNA, as seen in case of other dithiocarbamates (Gonzalez et al., 2003).
Histopathology of kidney tissues shows normal cellular architecture in control group but a progressive damage in renal parenchyma was observed in thiram treated rats. At 100 and 250 mg/kg body weight doses of thiram, degeneration of tubular epithelium was observed. At 500 mg/kg bw renal cast and at the highest dose of thiram (750 mg/kg bw) interstitial hemorrhage and saw-tooth appearance were observed in the cortical part of kidney. The damage to tubule cells led to the formation of tubular cast in conditions such as necrosis while interstitial hemorrhages are associated with acute kidney injury.

Conclusions
The ndings of the present study suggested that acute exposure of male rats to thiram induced nephrotoxicity as evidenced by induction of oxidative stress, reduction in antioxidant power (enzymatic and non-enzymatic), metabolic dysfunction, biochemical and histological alterations and DNA damage.
On the basis of our ndings, we may propose that studies should be carried out to determine kidney function in persons occupationally exposed to thiram.     Effect of thiram on the activities of (A) acid phosphatase and (B) total ATPase in cortex and medullary homogenate. Results are mean ± standard error of six different samples. *Signi cantly different from control at p < 0.05. ACP, acid phosphatase; ATPase; adenosine triphosphatase; COR, cortex; MED, medulla.

Figure 5
Effect of thiram on DNA fragmentation in rat kidney cells determined by diphenylamine assay. Results are mean ± standard error of six different samples. *Signi cantly different from control at p < 0.05. COR, cortex; MED, medulla.  green arrow (↑) in E indicates interstitial hemorrhage. In healthy control, the renal tubules and its epithelia are intact, with no tubular dilatation, cast or hemorrhage. The capillary tuft of the glomerulus is also intact. Kidneys from thiram treated animals at increasing doses show progressive damage in the renal parenchyma primarily in the tubules and also to some extent in the glomeruli. Tubular epithelia show progressive damage in the form of breaking of apical cytoplasm which is associated with cellular debris in the tubular lumen and quite often formation of renal casts. At higher doses of thiram, tubular epithelia