Oral administration of pentachlorophenol impairs antioxidant system, inhibits enzymes of brush border membrane, causes DNA damage and histological changes in rat intestine

Abstract

Pentachlorophenol (PCP) is a broad spectrum biocide that has many domestic and industrial applications. PCP enters the environment due to its wide use, especially as a wood preservative. Human exposure to PCP is through contaminated water and adulterated food products. PCP is highly toxic and is classified as class 2B or probable human carcinogen. In this study, we explored the effect of PCP on rat intestine. Adult rats were orally given different doses of PCP (25–150-mg/kg body weight/day) in corn oil for 5 days, whereas controls were given similar amount of corn oil. The rats were sacrificed 24 h after the last treatment. A marked increase in lipid peroxidation, carbonyl content, and hydrogen peroxide level was seen. The glutathione and sulfhydryl group content was decreased in all PCP treated groups. This strongly suggests the generation of reactive oxygen species (ROS) in the intestine. PCP administration suppressed carbohydrate metabolism, inhibited enzymes of brush border membrane (BBM), and antioxidant defense system. It also led to increase in DNA damage, which was evident from comet assay, DNA-protein cross-linking, and DNA fragmentation. Histological studies supported the biochemical results showing marked dose-dependent tissue damage in intestines from PCP treated animals. This study reports for the first time that oral administration of PCP induces ROS, impairs the antioxidant system, damages DNA, and alters the enzyme activities of BBM and metabolic pathways in rat intestine.

Introduction

Since the last 3 decades, the widespread discharge of pesticides in the environment has led to worldwide concern about their toxic effects. Pentachlorophenol (PCP) is a stable, noncorrosive compound used as fungicide, biocide, pesticide, and general disinfectant. PCP is used as preservative of timber, paper, paints, glues, textiles, and leather. It acts as antifungal and antialgal agent in aqueous systems and also has various agricultural applications.¹ Despite its usefulness, PCP poses potential risk to food safety, the environment and all living things. PCP is considered as priority pollutant in the environment and its generalized use has led to water contamination. Such indiscriminate use has resulted in considerable human exposure to PCP² leading to its bioaccumulation in the intestine, kidney, liver, and adipose tissue. There are several reports of PCP toxicity in humans and animals, sometimes with fatal outcomes.^1,3 PCP poisoning has no antidote and acute, high-level PCP exposure through any route can be lethal. A number of deaths have been attributed to PCP poisoning in occupational and nonoccupational settings.⁴ The diagnosis of PCP poisoning is difficult because of nonspecific signs and symptoms. Severe PCP poisoning results in organ system failure, hyperthermia, and death. A series of case reports has suggested risk of aplastic anemia and other nonmalignant hematological problems in PCP-exposed persons. Epidemiologic data on human carcinogenicity indicate the occurrence of soft tissue sarcoma while lymphoma is also reported.¹ PCP is, therefore, classified as Class 2B or probable human carcinogen.

PCP at millimolar level has been reported in surface water and ground water in industrial zones (0.7 mM) and also in industrial waste (0.5 mM).¹ PCP production workers at timber mills are estimated to inhale ~10.5–154 mg PCP/day and absorb ~35 mg PCP/day through dermal exposure.^5,6 In rhesus monkeys orally administered [¹⁴C]-labelled-PCP was monitored after 15 days and the largest fractions of radioactivity were obtained in liver, small intestine, and large intestine (1%, 5%, and 2%, respectively).¹ Autopsy reports of fatal cases of PCP poisoning in humans have shown that the maximum concentration of PCP was found in the bile and renal tissue, with lower concentrations in the lung, liver, and blood.^7,8 Binding of PCP (95–99.6%) to plasma proteins results in long half-life of PCP in humans.¹ Workers involved in PCP-utilizing industries and treated log homes are chronically exposed to PCP directly, and the highest concentration of PCP reported in liver was 59–62 mg/kg, in kidney 41–84 mg/kg and in blood 53–96 mg/L.^1,9 PCP elevates β-glucuronidase activity and inhibits nitroreductase enzyme in the small and large intestine, which transforms PCP into xenobiotic metabolites and also increases the formation of DNA-adducts.^10,11 During metabolism in liver of rodents and humans, PCP undergoes oxidative dechlorination to yield tetrachlorohydroquinone (TCHQ), semiquinone,¹² and H₂O₂. Under certain physiological conditions TCHQ and its corresponding semiquinone radical enter the autoxidation and reduction cycle in presence of oxygen to produce superoxide radicals. Thus, PCP metabolism could be a major reason for the generation of ROS in cells. PCP uncouples mitochondrial oxidative phosphorylation¹³ and produces ROS, which damage DNA and cell components.¹⁴

Intestine is the primary target after oral ingestion of any xenobiotic through contaminated food and/or water. Environmental contaminants and genotoxic metabolites like PCP enter the gastrointestinal tract via contaminated food, water, and air and many have been shown to be tumor initiators, promoters, and carcinogens. After exposure, PCP is readily absorbed from the intestine and then distributed to vital organs. Xenobiotics greatly affect the hydrolytic enzymes in the brush border membrane (BBM) that lines the intestinal mucosa and alter its functions like nutrient digestion and transport. Although, several studies have reported PCP toxicity in different tissues, there is none on its effect on the intestine. This study aimed to analyze the damaging effects of sublethal doses of PCP in rat intestine in detail. We show that oral administration of PCP impairs the antioxidant system, inhibits the enzymes of BBM, and pathways of glucose metabolism in rat intestine. In addition, DNA damage, DNA-protein cross-linking, and histological changes were also found in the intestine of male rats.

Material and methods

Materials

Chemicals: Pentachlorophenol (97% purity) was purchased from Sigma-Aldrich, United States. All other chemicals of analytical grade were obtained from Himedia (Mumbai, India), Sisco Research Laboratory (Mumbai, India), or Sigma-Aldrich (United States).

Animals and diet: Adult, male albino Wistar rats weighing 120–150 g were purchased from National Institute of Biologicals, NOIDA, Uttar Pradesh, India (CPCSEA reg. no. 824/GO/RBiBT/S/04/CPCSEA). They were fed standard rat pellet diet obtained from Aashirwaad Industries, Chandigarh, India.

Experimental protocol

Animals were housed under standardized condition for 1 week on rat pellet diet and water ad libitum. Rats were divided into 1 control and 4 PCP treated groups, with 5 animals in each group. PCP was dissolved in corn oil and doses of 25, 50, 100, and 150-mg/kg body weight were given to rats once a day for 5 consecutive days by oral gavage. Control rats were given corn oil by gavage. These doses were less than the reported oral LD₅₀ value of PCP in rats i.e. 211 mg/kg body weight.¹⁵

All animals were sacrificed 24 h after the last PCP treatment, under local anesthesia. The peritoneum cavity was opened and the entire small intestine was taken out and flushed with ice cold saline. The intestine was cut open longitudinally, mucosal layer was carefully scraped with glass slide and scrapings used to prepare homogenates and brush border membrane vesicles (BBMV). A small piece of intestine was preserved in RPMI for DNA damage studies (for comet assay) or homogenized in suitable buffer.

Preparation of mucosal homogenates and BBM vesicles

Mucosal scrapings were homogenized in 2-mM Tris–HCl, 50-mM mannitol buffer, and pH 7.0, to prepare a 10% (w/v) homogenate by giving 6 pulses of 35 s each using glass Teflon homogenizer. Samples (crude) were distributed in aliquots and kept at −80°C to analyze the nonenzymatic parameters. After centrifuging the remaining homogenates at 2,000 rpm (2K sample) and 5,000 rpm (5K sample) for 12–15 min at 4°C, the supernatants were preserved at −80°C to be later analyzed for various enzymatic activities. To the crude homogenates was added 10-mM CaCl₂ and BBM vesicles were isolated, exactly as done by.¹⁶ The protein content in BBM vesicles and homogenates was quantified by the method of Lowry.¹⁷

B‌BM enzymes

BBM enzyme activities were assayed in isolated BBM vesicles and 2K intestinal homogenates. Alkaline phosphatase (ALP), leucine aminopeptidase (LAP), and γ-glutamyl transferase (GGT) activities were determined by using p-nitrophenyl phosphate, l-leucyl p-nitroanilide, and γ-glutamyl p-nitroanilide as substrates, respectively.^18,19,20 Sucrase activity was measured from the enzymatic hydrolysis of sucrose to produce reducing sugars, which react with 3,5-dinitrosalicylic acid to yield a colored adduct.²¹

Nonenzymatic parameters of oxidative stress

Nonenzymatic markers of oxidative stress were measured in crude intestinal homogenates. Malondialdehyde (MDA) formation was determined as a measure of lipid peroxidation (LPO) from its reaction with color agent thiobarbituric acid that gives a pink complex.²² Carbonyl group content served as an index of protein oxidation and was measured from their reaction with 2,4-dinitrophenylhydrazine.²³ Reduced glutathione (GSH) levels were quantified in protein free tissue homogenates using 5,5′-dithiobisnitrobenzoic acid (DTNB), as described by Beutler.²⁴ The sulfhydryl (–SH) groups react with DTNB to give yellow thionitrobenzoate, which absorbs at 412 nm.²⁵ Hydrogen peroxide (H₂O₂) concentration was measured using FOX reagent (ferrous ammonium sulphate-xylenol orange); 0.1-M sorbitol was added as color enhancer.²⁶

Antioxidant status

The antioxidant (AO) power was determined in crude homogenates. In ferric reducing antioxidant power (FRAP), 0.1-mL intestinal homogenate was added to 1.5-mL FRAP reagent (containing 2,4,6-tris(2-pyridyl)-s-triazine and FeCl₃ in sodium acetate buffer, pH 3.6) and kept at room temperature for 5 min. The absorbance of blue–purple product formed was recorded at 593 nm.²⁷ K₃Fe(CN)₆ reduction assay was done as described by Yen and Chen.²⁸ In cupric reducing antioxidant capacity (CUPRAC) assay, AOs reduce Cu²⁺ to Cu⁺, which then gives a colored product with neocuproine.²⁹ In the phosphomolybdenum green assay, AOs in hemolysates convert Mo⁶⁺ to Mo⁵⁺; the latter forms a green colored phosphate-Mo⁵⁺ complex.³⁰ DPPH (2,2-diphenyl-1-picrylhydrazyl) assay was performed by incubating 0.1-mL mucosal homogenate with 0.5 mL of 100-μM DPPH solution in methanol and 0.4-mL of 10-mM sodium phosphate buffer, pH 7.4. The samples were kept at room temperature in dark for 30 min. After centrifugation at 12,000 rpm for 12 min, the absorbance of supernatants was recorded at 517 nm.³¹

The activities of AO enzymes responsible for free radical and ROS scavenging were assayed in 5K intestinal homogenates. Catalase activity was monitored from the enzymatic decomposition of H₂O₂ to H₂O at 240 nm³² and superoxide dismutase (SOD) was assayed from the enzyme catalyzed inhibition of pyrogallol auto-oxidation.³³ Glutathione reductase (GR) activity was determined from the conversion of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to NADP⁺ and following the decrease in absorbance at 340 nm.³⁴ Thioredoxin reductase (TR) activity was determined from the cleavage of DTNB to yield the yellow thionitrobenzoate anion in the presence of NADPH.³⁵ Glutathione peroxidase (GPx) activity was measured from the decrease in absorbance at 340 nm upon conversion of NADPH to NADP⁺, as described by Flohe and Gunzler.³⁶ Glutathione S-transferase (GST) activity was assayed by using 1-chloro-2,4-dinitrobenzene and GSH as substrates.³⁷

Membrane bound enzymes

Membrane bound enzymes were assayed in 2K samples. Total ATPase and Na⁺K⁺ATPase activities were quantified from the release of inorganic phosphate (Pi), upon cleavage of ATP to ADP and inorganic phosphate, in absence and presence of ouabain. The inorganic phosphate gives a blue complex with Taussky and Shorr reagent.³⁸

Enzymes of carbohydrate metabolism

The specific activities of metabolic enzymes were measured spectrophotometrically in 2K intestinal homogenates. Lactate dehydrogenase (LDH) was assayed from the conversion of reduced nicotinamide adenine dinucleotide (NADH) to NAD⁺ in presence of sodium pyruvate.³⁹ The activities of malate dehydrogenase (MDH), glucose 6-phosphatase (G6Pase), fructose 1,6-bisphosphatase (F6Pase), and malic enzyme (ME) were determined as described by Khundmiri.³⁹ Hexokinase (HK) was assayed by the method of Crane and Sols.⁴⁰ Glucose 6-phosphate dehydrogenase (G6PD) was measured by following the conversion of NADP⁺ to NADPH at 340 nm using glucose 6-phosphate as substrate.⁴¹ Acid phosphatase (ACP) was assayed from the yellow color produced upon cleavage of p-nitrophenyl phosphate to p-nitrophenol, at pH 4.5.⁴²

DNA damage studies

(i) Comet assay- single cell gel electrophoresis

The comet assay was performed as described by Singh⁴³ with some changes. The suspension (0.1 mL) of intestinal mucosa, prepared in RPMI medium, was added to 100 μL of 1% low-melting-agarose and immediately layered on agarose pre-coated slides. Another layer of agarose was put on the slide and allowed to solidify at 4°C. The further protocol was followed as described by Maheshwari and Mahmood.⁴⁴ After staining of DNA with ethidium bromide, the cells were scored under a CX41 fluorescent microscope attached with an image analysis system, Komet 5.5, Kinetic Imaging, Liverpool, United Kingdom. The comets from 100 cells (50 from each replicate slide) per sample were scored at a magnification of 100× and comet tail-lengths were recorded.

(ii) DNA fragmentation (DF) and DNA-protein cross-linking (DPC)

Diphenylamine method of Burton⁴⁵ was followed to determine DNA fragmentation and the results are reported as percent of fragmented DNA to total DNA. After homogenizing the intestinal mucosal tissue in Tris buffer (20-mM Tris–HCl, 2% SDS, 20-mM EDTA, pH 7.5), the DPCs were analyzed by the K⁺-SDS assay, exactly as done by Zhitkovich and Costa.⁴⁶

Histopathology

A 2–3-cm long section of the duodenal portion of intestine was preserved in 10% formalin solution, processed and fixed in paraffin blocks. A 5-μm thin microscopic section was cut from the paraffin block and layered on glass slide. The cells were stained with hematoxylin and eosin (H & E) and visualized under a microscope at 100-fold magnification.⁴⁷

Statistical analysis

All experiments were done on 5 different animals in each group. Results obtained are given as mean ± standard error of mean. Statistical analyses were performed by software Origin Pro 8 (United States) using analysis of variance (ANOVA) 1-way analysis and assessed by Post-Hoc test (Bonferroni comparison test). The results of PCP treated groups were compared with the untreated control group and P ≤ 0.05 was considered as statistically significant.

Results

B‌BM enzymes

BBM enzyme activities were measured in isolated BBM vesicles and intestinal mucosal homogenates. Oral administration of PCP greatly lowered BBM enzyme activities in BBM vesicles. The percent remaining activity seen in the highest PCP dose group (150-mg/kg body weight) was—ALP 38.7%, LAP 39.7%, and GGTase 47%, when compared with the control group (Fig. 1A). A similar trend was observed in mucosal homogenates with BBM enzyme activities declining in a PCP dose-dependent manner. The percent activity seen in highest PCP dose group was—ALP 48.7%, LAP 41%, and GGTase 30%. The activity of intestinal BBM marker enzyme, sucrase was greatly decreased to 33% in isolated BBM vesicles and also in mucosal homogenates (Fig. 1B).

Fig. 1.

Specific activities of BBM marker enzymes were determined in A) isolated rat intestinal BBM vesicles (reported in μmoles/mg protein/h) and in B) rat intestinal homogenates (reported in nmoles/mg protein/h).

The bar graphs are mean ± standard error of 5 different preparations. Each bar also shows all the data points (•) obtained in the experiments. ^*Significantly different at P < 0.05 from control by 1-way ANOVA. Abbreviations: ALP, alkaline phosphatase; LAP, leucine aminopeptidase; C, control; GGT, γ-glutamyl transferase, PCP, pentachlorophenol.

Nonenzymatic oxidative stress markers

Nonenzymatic markers of cellular oxidative stress were measured in mucosal homogenates prepared from control and PCP treated animals. Results showed that PCP dose-dependently changed the nonenzymatic parameters in rat intestine (Table 1). H₂O₂ level was increased to 2.5-fold in comparison with control. Protein oxidation incorporates carbonyl groups in amino acid side chains and was enhanced to 3.3-fold in the highest PCP dose group, when compared with control. The GSH and total SH levels were decreased to 32% and 51%, respectively, which will lower the reducing ability of mucosal cells (Table 1). LPO was increased 2.6-fold in the highest dose group (150 mg/kg bw) when compared with control animals (Table 1).

Table 1.

PCP-induced changes in non-enzymatic parameters of oxidative stress in rat intestine.

		PCP (mg/kg body weight)
	Control	25	50	100	150
GSH	7.07 ± 0.94	5.51 ± 0.76*	4.31 ± 0.58*	3.31 ± 0.42*	2.28 ± 0.35*
Total-SH	395.35 ± 46.34	317.73 ± 37.16*	275.65 ± 31.44*	242.83 ± 29.24*	205.05 ± 23.67*
PO	68.71 ± 8.31	96.13 ± 12.53*	115.98 ± 16.35*	142.26 ± 20.11*	223.98 ± 25.43*
LPO	2.44 ± 0.31	3.81 ± 0.48*	4.14 ± 0.52*	5.82 ± 0.79*	6.35 ± 0.91*
H2O2	12.61 ± 1.52	16.41 ± 1.86*	18.00 ± 2.03*	22.82 ± 3.18*	31.42 ± 3.23*

GSH, total-SH, PO, LPO, and H₂O₂ levels are in nmoles/mg protein.

Results are mean ± standard error of 5 different preparations.

^*Significantly different at P < 0.05 from control by 1-way ANOVA.

Abbreviations: GSH, reduced glutathione; SH, sulfhydryl group; PO, protein oxidation; LPO, lipid peroxidation; H₂O₂, hydrogen peroxide.

Antioxidant status

To examine the role of AO system in PCP-induced toxicity, the effect of PCP on AO power of mucosal cells was studied. AOs in sample quench free radicals or reduce metal ions to their lower oxidation state by donating electron or H atom. The reducing capacity of cells was determined by FRAP, K₃Fe(CN)₆, PMG, CUPRAC, and DPPH assays. These assays showed the AO capacity of mucosal cells was lowered to 62% in the highest PCP dose group (150-mg/kg bw) as compared with control animals (Table 2).

Table 2.

PCP-induced changes in the antioxidant power of rat intestine.

		PCP (mg/kg body weight)
	Control	25	50	100	150
FRAP	435.2 ± 56.17	427.3 ± 48.30	394.5 ± 41.39*	353.8 ± 38.61*	288.5 ± 30.58*
K3Fe(CN)6	101.3 ± 13.67	96.5 ± 11.16	92.1 ± 10.53	85.1 ± 9.50*	68.3 ± 7.66*
PMG	95.1 ± 12.10	87.7 ± 10.08	71.4 ± 7.25*	64.7 ± 7.18*	50.7 ± 6.15*
CUPRAC	219.3 ± 31.50	200.2 ± 22.26	177.7 ± 20.13*	165.0 ± 18.30*	132.0 ± 15.22*
DPPH	94.4 ± 11.17	91.0 ± 10.02	85.8 ± 9.50*	77.4 ± 8.84*	65.0 ± 7.95*

FRAP, K₃Fe(CN)₆, PMG and CUPRAC are in nmoles/mg protein and DPPH in % quenching.

Results are mean ± standard error of 5 different preparations.

^*Significantly different at P < 0.05 from control by 1-way ANOVA.

Abbreviations: FRAP, ferric reducing antioxidant power; K₃Fe(CN)₆, potassium ferricyanide reduction assay; PMG, phosphomolybdenum green; CUPRAC, cupric reducing antioxidant capacity; DPPH, 2,2-diphenyl-1-picrylhydrazyl assay.

ROS generated by xenobiotics play an important role in cell injury in various tissues. AO enzymes provide major cellular defense against ROS. A remarkable decrease was observed in the activities of AO enzymes in a PCP dose-dependent manner. The activity in the highest PCP dose group, as compared with control group, was—CAT 50%, SOD 52%, GPx 50%, TR 50%, and GR 64%. GST activity was increased to 1.9-fold in comparison with control value (Table 3).

Table 3.

PCP-induced changes in the activities of major antioxidant enzymes in rat intestine.

		PCP (mg/kg body weight)
	Control	25	50	100	150
Catalase	62.16 ± 8.61	53.12 ± 6.64*	45.47 ± 5.81*	38.59 ± 4.56*	30.93 ± 3.48*
SOD	80.22 ± 10.67	73.13 ± 8.13*	65.31 ± 7.47*	57.74 ± 6.92*	41.67 ± 5.60*
GPx	142.16 ± 16.08	118.11 ± 13.36*	110.59 ± 12.61*	92.71 ± 10.19*	71.14 ± 8.37*
GR	29.99 ± 3.80	27.85 ± 3.17	24.40 ± 2.88*	22.02 ± 2.72*	19.36 ± 2.09*
TR	65.90 ± 8.79	58.51 ± 6.15	49.98 ± 5.36*	40.01 ± 4.71*	32.69 ± 4.82*
GST	13.74 ± 1.64	17.04 ± 2.57*	19.43 ± 2.81*	22.32 ± 3.07*	26.12 ± 3.72*

Specific activity of SOD is in units/mg protein. Catalase, GPx, GR, TR, and GST are in nmoles/mg protein/min.

Results are mean ± standard error of 5 different preparations.

^*Significantly different at P < 0.05 from control by 1-way ANOVA.

Abbreviations: SOD, superoxide dismutase; GPx, glutathione peroxidase; GR, glutathione reductase; TR, thioredoxin reductase; GST, glutathione S-transferase.

Membrane bound enzymes

Total ATPase and Na⁺K⁺ATPase were inhibited with increasing dose of PCP. The enzyme activity in the highest PCP dose group (150-mg/kg bw) as compared with control was—total ATPase, 58% and Na⁺K⁺ATPase, 61% (Table 4).

Table 4.

PCP-induced changes in the ATPase activities in rat intestine.

		PCP (mg/kg body weight)
	Control	25	50	100	150
Total ATPase	4.34 ± 0.482	4.16 ± 0.501	3.82 ± 0.410*	3.38 ± 0.364*	2.53 ± 0.307*
Na+K+ ATPase	204.39 ± 24.81	198.89 ± 22.74	162.61 ± 20.26*	151.27 ± 17.67*	125.38 ± 16.56*

Specific activity of total ATPase is in μmoles/mg protein/h and Na⁺K⁺ ATPase in nmoles/mg protein/h.

Results are mean ± standard error of 5 different preparations.

^*Significantly different at P < 0.05 from control by 1-way ANOVA.

Abbreviation: ATPase, adenosine triphosphatase.

Enzymes of carbohydrate metabolism

Studies based on cellular energetics reveals the role of metabolic pathways in elucidating cell fate and response to injury. Enzymes of glucose metabolic pathways and NADPH production were assayed in mucosal homogenates to ascertain the effect of PCP on energy metabolism. PCP administration decreased the enzyme activities dose-dependently and the activity in the highest dose group, relative to control, was—HK 48%, PK 56%, MDH 48%, ACP 58%, G6Pase 48%, F6Pase 44%, and G6PD 47%. However, the enzyme activity was significantly increased in case of LDH (2.4-fold) and ME (3-fold) in comparison with control group (Table 5).

Table 5.

PCP-induced changes in the activities of metabolic enzymes in rat intestine.

		PCP (mg/kg body weight)
	Control	25	50	100	150
HK	10.65 ± 1.92	8.75 ± 1.05	8.97 ± 0.98	7.55 ± 0.86*	5.09 ± 0.75*
PK	65.36 ± 7.55	52.96 ± 6.34	47.30 ± 5.92*	42.64 ± 5.24*	36.94 ± 4.07*
LDH	86.41 ± 10.82	118.19 ± 13.03	133.94 ± 15.72*	173.09 ± 19.13*	207.99 ± 25.10*
G6PD	272.73 ± 31.51	238.55 ± 26.21	204.56 ± 22.61*	172.79 ± 19.81*	127.43 ± 18.33*
MDH	302.96 ± 33.17	267.25 ± 29.21*	218.17 ± 25.55*	178.96 ± 20.68*	146.35 ± 16.52*
ME	19.32 ± 2.84	24.40 ± 3.02*	32.66 ± 3.91*	42.19 ± 4.77*	58.09 ± 6.81*
FBPase	504.47 ± 55.61	468.07 ± 51.27	389.33 ± 46.64*	322.30 ± 40.33*	221.51 ± 30.15*
G6Pase	547.85 ± 60.36	508.62 ± 57.61	431.11 ± 50.42*	367.54 ± 40.57*	261.83 ± 31.34*
ACP	33.64 ± 5.23	31.66 ± 3.91	27.57 ± 3.05*	24.23 ± 3.21*	19.39 ± 2.02*

Specific activity of HK is in μmoles/mg protein/min, LDH, G6PD, ACP, MDH, ME, FBPase, and G6Pase are in nmoles/mg protein/min.

Results are mean ± standard error of 5 different preparations.

^*Significantly different at P < 0.05 from control by 1-way ANOVA.

Abbreviations: HK, hexokinase; PK, pyruvate kinase; LDH, lactate dehydrogenase; G6PD, glucose 6-phosphate dehydrogenase; MDA, malate dehydrogenase; ME, malic enzyme; F6Pase, fructose 1,6-bisphosphatase; G6Pase, glucose 6-phosphatase; ACP, acid phosphatase.

DNA damage

The diphenylamine assay showed that PCP induces DNA fragmentation in a PCP dose-dependent manner in rat mucosa. The degradation of DNA was increased and in the highest PCP treated animals it was 3.8-fold of the control value (Fig. 2). Further confirmation of this observation came from the comet assay (Fig. 3). Comet tail-length is linked to fragment size and proportional to the extent of single stranded cleavages and alkali-labile sites. The comet tail-length was increased in a dose-responsive manner in PCP-administered groups in comparison with untreated control animals. This signifies strand scission and DNA degradation. The K⁺-SDS assay clearly showed that PCP increased DNA-protein cross-linking (DPC) in rats. The percentage of DPC formation in the highest dose group was 2.33 times the control value (Table 6).

Fig. 2.

DNA fragmentation in intestinal mucosal cells of control and PCP-treated rats determined by diphenylamine assay. The bar graphs are mean ± standard error of 5 different preparations. Each bar also shows all the data points (•) obtained in the experiments. ^*Significantly different at P < 0.05 from control by 1-way ANOVA. Abbreviations: DF, DNA fragmentation; PCP, pentachlorophenol.

Fig. 3.

DNA damage in intestinal mucosal cells studied by the comet assay. A) DNA of cells was visualized under a fluorescent microscope from a) control and b)–e) 25, 50, 100, and 150 mg PCP/kg body weight, respectively. B) Comet tail-lengths. The bar graphs are mean ± standard error of 5 different preparations. Each bar also shows all the data points (•) obtained in the experiments. ^*Significantly different at P < 0.05 from control by 1-way ANOVA. Abbreviation: PCP, pentachlorophenol.

Table 6.

Effect of PCP on DNA-protein cross-linking in intestine of rats.

		PCP (mg/kg body weight)
	Control	25	50	100	150
a DNA-protein crosslinks %	2.55 ± 0.28	2.81 ± 0.33	3.69 ± 0.41*	4.40 ± 0.56*	5.95 ± 0.72*
b DNA-protein crosslinks coefficient	1.0	1.10	1.45	1.72	2.33

Results are mean ± standard error of 5 different preparations.

^*Significantly different at P < 0.05 from control by 1-way ANOVA.

^aDNA-protein crosslinks / total DNA.

^bDNA-protein crosslinks (%) in PCP treated animals / DNA-protein crosslinks (%) in control animals.

Histopathology

Intestine prepares and absorbs nutrients and serves as the first line of defense against toxic insult through oral and other routes of exposure. Upon examination, intestines in PCP treated rats appeared to be dehydrated, twisted, tough, and leathery (Fig. 4A). Microscopic examination of tissues at 100X magnification showed marked damage in the intestinal villi of PCP treated animals (Fig. 4B).

Fig. 4.

A) Morphological analysis of intestine from control and PCP treated (25, 50, 100, and 150 mg/kg body weight) animals. B) Histopathological analysis of rat intestine. Representative photomicrograph from rat intestine showing intestinal villi from control A) and those from the PCP treated groups (B, C, D, and E with PCP doses of 25, 50, 100, and 150-mg/kg body weight, respectively). Black arrows indicate villi, yellow arrows indicate intestinal glands (crypts), and green arrows indicate the connective tissue support of the villi (lamina propria). In control group the intestinal villi show normal architecture in terms of lining epithelium, close adherence between epithelium and the lamina propria, and the villus to crypt ratio, which is within normal limits. Group B shows appearance of a gap between epithelium and lamina propria but other parameters were unaltered. Group C shows increased gap between epithelium and lamina propria along with damage to lining epithelial cells; group D shows gap, damage to epithelial cells, and shedding of epithelial lining of villi, whereas group E shows naked lamina propria because of complete loss of lining epithelium of villi. The lining epithelium of glands (crypts) remains conspicuously intact in all treated groups. Initial magnification is 100X with H & E staining.

Discussion

Industrialization has resulted in emissions and ubiquitous dispersal of persistent organochlorines in local and global environments leading to bioaccumulation in organisms. PCP is a commonly used organochlorine compound in agricultural pesticides that causes ROS generation, lesions as well as cancer in farmers and commercial pesticide users. Commercially available fungicides Basilit and Creosote contain 2% PCP, fungicide and rodenticide Penwar 1–5 (EPA reg. no. 7234–7) contains 25.6% PCP, antimicrobial Dowicide-5 contains 85% PCP, and wood preservative Chlorophen contains 22.4% PCP.⁴⁸ Oral exposure to PCP through any route results in its quick absorption through the gastrointestinal tract, from where it enters the closely associated circulatory system and made available to other tissues. This work was undertaken to investigate the multifaceted damaging effects of PCP on DNA integrity, antioxidant defense, enzymes of BBM, glucose metabolic pathway, lysosomes, and histology of small intestine.

The intestinal epithelial cells lining the lumen are highly susceptible to oxidant attack and damage due to constant exposure to xenobiotics.^49,50 To overcome this cellular defense systems are present that are essential for protection from oxidative stress and to attenuate oxidative injury. After absorption in gastrointestinal tract PCP is transformed by conjugation, hydrolytic, and reductive dechlorination in humans and animals.⁵¹ Studies have reported that PCP undergoes redox cycling and is converted to highly toxic TCHQ and TCBQ (tetrachloro-p-benzoquinone). Autoxidation of TCHQ and/or the reduction of TCBQ by NADH produce semiquinone radicals. These radicals react with molecular O₂ to form H₂O₂ and superoxide, which are later activated by transition metals to cause cellular damage.⁵² This may lead to increased H₂O₂ level in PCP-administered rats, in a dose-dependent manner. Generation of free radicals and increased H₂O₂ level lowered the AO power, which will result in oxidative damage of cell components. This decrease in AO power could be due to reduced levels of GSH, a thiol containing tripeptide that is a major cellular AO, and lower activities of AO enzymes. The specific activity of antioxidant enzymes was decreased dose-dependently upon PCP administration. ROS and free radicals have been shown to inhibit SOD, CAT, GPx, and oxidatively modify TR and GR.^53,54 SOD, CAT, and GPx contribute to the first line of AO defense mechanism. In PCP-administered rats inhibition of these AO enzymes will further increases the production of H₂O₂, a non-radical ROS_. GR catalyzes the NADPH-dependent reduction of GSSG to GSH. Lowered GR activity will increase the GSSG: GSH ratio, which serves as a marker of cellular toxicity. TR regulates cellular redox balance by mitigating the damage caused by ROS and also keeps proteins in reduced state. Oral administration of PCP inhibited the TR activity probably by modifying its cysteine residues at the catalytic site.⁵⁵ An increase in GST activity might be an adaptive response of mucosal cells to detoxify xenobiotics by conjugating them with GSH. Impairment of the enzymatic and nonenzymatic AO defense systems of the cells makes the tissue vulnerable to oxidative damage and may be responsible for the intestinal toxicity of PCP. These results suggest that the toxic effect of PCP is mediated by enhanced generation of ROS and induction of oxidative stress condition.⁵⁶ Elevated level of ROS damages cellular macromolecules including proteins, lipids, and mitochondrial and nuclear DNA. The end result of this cascade is cellular dysfunction and/or death.

ROS and reactive nitrogen species cause oxidation of matrix and membrane proteins leading to electrolyte imbalance, cellular dysfunction, membrane impermeability, and matrix protein degradation.^57,58 ROS oxidize side chain amino groups and introduce carbonyl groups that are commonly used as marker of protein oxidation. A significant increase in protein oxidation was seen in highest PCP dose group. PCP lowered the thiol status of intestinal mucosal cells (GSH and total-SH) that compromised an important line of defense against oxidative damage. Endogenous AOs such as GSH can react with and quench ROS, thus restoring cellular function and homeostasis. PCP either forms conjugate with GSH or cleaves glycine and glutamate to give cysteine conjugates.⁵⁹ Declined GSH level also affects the activities of GSH-dependent enzymes (GR, GPx, and GST) that use GSH as substrate. MDA is a toxic end product of LPO that is produced upon attack of free radicals on unsaturated fatty acids in membrane lipids. The radical extracts a hydrogen atom from lipids to form lipid radicals and initiates oxygen-mediated chain reaction that riddles the membrane with lipid hydroperoxides. Increased LPO causes oxidation of collagen, resulting in modification, fragmentation, aggregation, and conformational changes in protein.⁶⁰ This eventually leads to alterations in intestinal tissue functioning and senescence.

Intestine BBM possesses transport systems for amino acids, secretes enzymes involved in carbohydrate metabolism, absorption, and transport of ions.^61,62,63 Since the intestinal mucosa is the major functional site and primary target of toxicants like PCP, the BBM integrity was determined from the activity of biomarker enzymes in isolated BBM vesicles and in mucosal homogenates. All 4 BBM marker enzymes were inhibited, both in isolated membrane vesicles and homogenates from PCP treated animals. Lowered ALP can compromise gut homeostasis, detoxification of lipopolysaccharides and dephosphorylation of proinflammatory nucleotides. It also causes intestinal inflammation, dysbiosis, gut microbes translocation leading to systemic inflammation.⁶⁴ Decrease in LAP minimizes its role in protein degradation.⁶⁵ GGTase is a cell surface enzyme that primarily metabolizes extracellular GSH into its precursor amino acids allowing them to be assimilated and reutilized for intracellular GSH synthesis and also minimizes oxidative stress. Inhibition of GGTase will lower cysteine levels in the body,⁶⁶ which will prevent incorporation of cysteine in intracellular GSH synthesis resulting in rapid decrease in GSH level.⁶⁷ Sucrase is secreted in the small intestine on the brush border, and its activity was significantly decreased. The BBM enzyme inhibition can be attributed to certain factors (i) PCP-induced oxidation of membrane proteins and lipids causes disruption of membrane integrity leading to loss of enzyme molecules from the epithelial lining in intestine. (ii) ROS can either directly oxidize enzyme molecules or modify enzyme active site and inhibit enzyme activity. (iii) PCP acts as an uncompetitive inhibitor and inhibits enzyme activity either by binding to the enzyme-substrate complex or by interfering with the hydrolysis of the phosphoenzyme intermediate.⁶⁸ Thus direct binding of PCP or its metabolites could also have led to inhibition of BBM enzymes.

ATPases are highly conserved integral membrane proteins located in the basolateral membrane of enterocytes that regulate intracellular osmotic balance and membrane potential gradient.⁶⁹ Increased LPO and protein oxidation causes disintegration of membrane structure resulting in leakage, loss, and dysfunction of membrane bound enzymes. Oral administration of PCP inhibited total ATPase and Na⁺K⁺ATPase, which will result in derangement of the key function of enterocytes i.e. active absorption of nutrients and maintenance of solutes and ions in the body. PCP is an active uncoupler of oxidative phosphorylation. Like other chlorophenols, PCP binds to mitochondrial proteins and inhibits mitochondrial ATPase activity.^70,71 PCP prevents incorporation of inorganic phosphate into ATP, preventing the formation and breakdown of ATP, which lowers the release of energy to the cell.^72,73 Thus, the cells continue to respire but are soon depleted of ATP necessary for growth.

Small intestine maintains glucose homeostasis by absorbing, metabolizing, and distributing dietary glucose to tissues. Although we have not determined the actual rates of any pathway, the results showed that the specific activities of enzymes involved in glycolysis, TCA cycle, gluconeogenesis and HMP shunt were significantly altered by PCP treatment. PCP binds to the enzymic protein and blocks the reactions based on the high-energy intermediate that affects both oxidative and glycolytic phosphorylation. LDH catalyzes the anaerobic conversion of pyruvate to dead end product lactate and NAD⁺. MDH is a key enzyme in the central oxidative pathway that reversibly converts malate to oxaloacetate using NAD⁺ or NADP⁺ as a cofactor. Oxaloacetate is an intermediate of the tricarboxylic acid (TCA) cycle and produces ATP and NADH through the aerobic respiratory chain oxidation. Enhanced LDH and inhibition of MDH activity indicates a switch from aerobic energy production to anaerobic mode.⁷⁴ A decrease in HK activity will deprive the cells of energy. PK is an important enzyme in glycolytic pathway, BBM damage causes loss in PK activity, and its discharge in feces. PK in fecal matter serves as biomarker of inflammation and carcinogenesis in gastrointestinal mucosa.⁷⁵

Normally, gluconeogenesis occurs in the liver, but intestinal gluconeogenesis is important during conditions of hepatic damage and starvation.⁷⁶ G6Pase catalyzes the final step in gluconeogenesis, glycogenolysis, and regulates blood glucose homeostasis. G6Pase dephosphorylates glucose 6-phosphate to glucose, which is then exported from the cells via glucose transporter membrane proteins. F6Pase is a rate limiting enzyme involved in the anabolic pathways, gluconeogenesis, and Calvin cycle. PCP administration decreased G6Pase and F6Pase activities that can be due to oxidative modification and membrane disruption. Another reason can be inhibition of MDH leading to decrease in oxaloacetate, a precursor of gluconeogenesis and TCA cycle.

G6PD is an indispensable enzyme of the pentose phosphate pathway that generates major cellular reductant NADPH. ME catalyzes reversible oxidative decarboxylation to generate reducing equivalents. NADPH is critically important as it provides the reducing power that fuels the protein-based antioxidant systems and recycles oxidized glutathione. A PCP-dose-dependent decrease in G6PD activity will lower NADPH and make the cells more susceptible to oxidant attack. Increased ME activity in PCP treated groups was seen, which can be considered as an adaptive response to overcome the decrease in G6PD activity. Oral administration of PCP elicited significant decrease in the activity of ACP, a marker enzyme for lysosomes, which indicates oxidative damage to lysosomes along with BBM.

PCP and its metabolites are highly toxic and considered to be potential mutagenic and carcinogenic agent in humans.¹² They generate superoxide radicals, hydroxyl radicals (OH^•) and H₂O₂, which is activated by transition metals to cause DNA damage.⁵² PCP is a known hepatocarcinogen, single oral dose (80-mg/kg bw) increases the level of 8-OH-dG in rats, which is a significant marker of oxidative DNA damage.⁷⁷ A significant increase was seen in DNA strand breaks in intestinal mucosal cells determined by single cell gel electrophoresis (comet assay). DNA fragmentation and DNA-protein cross-linking was accelerated in rats upon oral administration of PCP. This DNA damage could be due to covalent binding of PCP and its metabolites to DNA or action of PCP-induced ROS to form DNA-adducts or strand scission in treated rats.⁷⁸

The presence of microvilli increases the surface area for absorption in the intestine. After oral administration of PCP for 5 days, in control rats the intestines were soft, flexible, and smooth, whereas in 150-mg/kg body weight group intestines were shrunken, brittle, and twisted. Photomicrograph of stained section of control rat intestine showed normal epithelium lining and close adherence between epithelium and the lamina propria. The villus to crypt ratio is within normal limits. In PCP treated groups the gap between epithelium and lamina propria, shedding of epithelial lining, damage to epithelial cells, and villi was increased dose-dependently. The epithelium lining of crypts glands remains conspicuously intact in all treated groups.

Conclusions

Oral administration of PCP causes severe toxicity and extensive structural damage to the small intestine. It increased oxidation of thiols, proteins, and lipids and elevated H₂O₂ level, which clearly indicates oxidative damage to enterocytes. Activities of enzymes of BBM, carbohydrate metabolism, and AO defense were greatly affected by PCP treatment. The enzymatic and nonenzymatic AO defense systems of the enterocytes were weakened, which will decrease their capacity to quench free radicals and ROS generated upon PCP treatment. DNA strand scission and DNA-protein cross-linking also occurred. Histopathological images clearly show visible damage caused by PCP treatment. All the effects were PCP dose-dependent. The study of such biochemical and molecular events taking place in the intestine will yield information that can be used to design methods to lower the damaging effects of environmental toxicants like PCP.

A general schematic representation of PCP-induced intestinal toxicity based on this study is shown in Fig. 5.

Fig. 5.

Schematic representation of PCP-induced generation of ROS in intestinal mucosal cells. PCP increases oxidation of membrane proteins and lipids leading to membrane damage that further promotes PCP influx. The enzymes of BBM were inhibited. PCP alters enzyme activities, lowers GSH and impairs antioxidant status of cells. The pathways of carbohydrate metabolism were inhibited. PCP damages DNA by promoting DNA-protein cross-linking, DNA fragmentation and strand scission. Abbreviations: PCP, pentachlorophenol; AO, antioxidants; BBM, brush border membrane; DPC, DNA-protein cross-linking; GSH, reduced glutathione; ROS, reactive oxygen species; RNS, reactive nitrogen species.

Animal ethical clearance

Ethical approval for animal experiments was obtained from the Institutional Animal Ethics Committee (IAEC) of the Department of Biochemistry, Faculty of Life Sciences, Aligarh Muslim University; the ethics approval number is 714/GO/Re/S/02/CPCSEA.

Funding

We gratefully acknowledge UGC-SAP-DRS-III, DST-PURSE-II, and DBT-BUILDER schemes for providing financial support to the Department of Biochemistry.

Conflict of interest

The authors declare no conflict of interest in this work.

Authors’ contributions

Nikhil Maheshwari took the responsibility of experimental work, data analysis, and manuscript writing. Riaz Mahmood did supervision, analysis, editing, and data curation. Aijaz Ahmed Khan was involved in histopathology.