Association of organochlorine pesticides with the mRNA expression of tumour necrosis factor-alpha (TNF-α) & cyclooxygenase-2 (COX-2) genes in idiopathic preterm birth.

Background & objectives: Preterm birth (PTB) is an important cause of prenatal death, neonatal morbidity and mortality and adult illness. Increased inflammation occurs in normal parturition, and inflammatory cytokines and oxidative stress are found to be higher in PTB cases. The present study was planned to investigate the association of organochlorine pesticides (OCPs) with mRNA expression of inflammatory pathway genes such as tumour necrosis factor-alpha (TNF-α) and cyclooxygenase-2 (COX-2) in preterm delivery (PTD) cases. Methods: Maternal blood samples of PTD (n=30) cases and equal number of term delivery (n=30) were collected at the time of labour. Women occupationally exposed to OCPs and other high risk factors such as anaemia, hypertension, bacterial vaginosis, renal and heart disease, diabetes, etc. were excluded. The OCP levels were estimated by gas chromatography, and mRNA expressions of TNF-α and COX-2 genes were analysed using real-time PCR (qPCR). Results: Significantly higher levels of β-HCH (beta-hexachlorocyclohexane, 95% CI=2.08-4.633, P=0.001), p’p’-DDE (para, para-dichlorodiphenyldichloroethylene, 95% CI=0.546-2.551, P=0.003), and o’p’-DDD (ortho, para-dichlorodiphenyldichloroethane, 95% CI=0.004-0.690, P=0.047) were observed in maternal blood of PTB cases as compared to term delivery. The mRNA expressions of COX-2 and TNF-α genes were 3.13 and 2.31 folds higher in PTB cases in comparison to term delivery. Linear positive correlations were observed between period of gestation (POG) and ΔCt of COX-2 and TNF-α genes. Interpretation & conclusions: Environmental factors such as OCPs may be associated with inflammatory events showing gene-environment interaction in PTB cases. Evaluating the molecular control of inflammation along with gene environment interaction may be used as a model to explore the aetiology of idiopathic PTB cases and may be considered for the prognosis of adverse reproductive outcomes.

Induction of oxidative stress and histopathological changes by sub-chronic doses of triazophos.

The effect of triazophos (O, O-diethyl O-1-phenyl-1 H-1, 2, 4-triazol-3-yl phosphorothioate), a widely used insecticide was studied on the induction of oxidative stress and histological alterations at sub-chronic doses in male albino rats. Oral administration of triazophos at concentrations of 1.64, 3.2 and 8.2 mg/kg body wt for 30 days produced dose as well as time-dependent increase in the lipid peroxidation (determined by malondialdehyde levels) and glutathione-S-transferase (GST) activity in serum with a concomitant decrease in ferric reducing ability of plasma (FRAP) and blood glutathione (GSH) content. Histopathological examination of liver of triazophos-treated rats showed significant and progressive degenerative changes as compared to control, which could be due to induction of oxidative stress. However, no significant histopathological changes were observed in spleen, kidney and brain at either dose of triazophos with respect to control. These results indicated that oral administration of triazophos was associated with enhanced lipid peroxidation and compromised antioxidant defence in rats in dose and time-dependent manner. Thus the present study demonstrated for the first time the role of oxidative stress as the important mechanism involved in the stimulation of hepatic histo-architectural alterations at sub-chronic doses of triazophos in rats.

Propoxur-induced acetylcholine esterase inhibition and impairment of cognitive function: Attenuation by Withania somnifera.

Propoxur (2-isopropoxyphenyl N-methylcarbamate) is widely used as an acaricide in agriculture and public health programs. Studies have shown that sub-chronic exposure to propoxur can cause oxidative stress and immuno-suppression in rats. Carbamates are also known to exhibit inhibitory effect on cholinesterase activity, which is directly related to their cholinergic effects. In the present study, the effect of Withania somnifera (Ashwagandha), a widely used herbal drug possessing anti-stress and immuno-modulatory properties was studied on propoxur-induced acetylcholine esterase inhibition and impairment of cognitive function in rats. Male Wistar rats were divided into four groups. Group I was treated with olive oil and served as control. Group II was administered orally with propoxur (10 mg/kg b.wt.) in olive oil, group III received a combination of propoxur (10 mg/kg b.wt.) and W. somnifera (100 mg/kg b.wt.) suspension and group IV W. somnifera (100 mg/kg b.wt.) only. All animals were treated for 30 days. Cognitive behaviour was assessed by transfer latency using elevated plus maze. Blood and brain acetylcholine esterase (AChE) activity was also assessed. Oral administration of propoxur (10 mg/kg b.wt.) resulted in a significant reduction of brain and blood AChE activity. A significant prolongation of the acquisition as well as retention transfer latency was observed in propoxur-treated rats. Oral treatment of W. somnifera exerts protective effect and attenuates AChE inhibition and cognitive impairment caused by sub-chronic exposure to propoxur.

Monitoring oxidative stress across worsening child pugh class of cirrhosis.

CONTEXT :Oxidative stress has been increasingly implicated in the pathogenesis and progression of cirrhosis. AIMS :We studied oxidative stress in patients with cirrhosis by measuring markers reflecting pro-oxidant (serum malondialdehyde-MDA) and antioxidant factors (RBC catalase-CAT, superoxide dismutase-SOD and blood reduced glutathione-GSH) factors. The level of oxidative stress was also assessed with respect to functional compromise of liver, as determined by Child Turcotte Pugh (CTP) scoring. DESIGN :Case-controlled retrospective study. MATERIALS AND METHODS :Twenty-three patients of cirrhosis along with 23 age and sex matched healthy controls were studied. Exclusion criteria were concurrent use of anti-oxidant drugs; co-existing diseases like DM, CKD; alcohol use, gastrointestinal bleed or blood transfusion within previous 2 weeks. Besides routine investigations, MDA, CAT, SOD and GSH levels were measured and compared with controls. STATISTICAL ANALYSIS :Continuous variables were recorded as mean +/- SD; ANOVA-f test, followed by Tukey's test, was used to evaluate the significance of difference (P < 0.05) among groups. RESULTS :Mean age of patients was 41.04 +/- 12.3 yrs. Patients showed a significant increase in MDA {control 3.31 +/- 0.25 (95% CI 3.21-3.41), Child B 6.30 +/- 0.4 (95% CI 6.03-6.53), Child C 8.05 +/- 0.66 (95% CI 7.29-8.81) nmol/l} and a significant decrease in levels of SOD {control 845.13 +/- 36.44 (95% CI 829.92-860.34), Child B 582.91 +/- 42.12 (95% CI 557.45-608.32), Child C 489.5 +/- 17.66 (95% CI 479.3-499.7) U/gm Hb}, CAT {controls 2.54 +/- 0.22 (95% CI 2.45-2.63), Child B 1.93 +/- 0.23 (95% CI 1.72-2.14), Child C 1.46 +/- 0.10 (95% CI 1.40-1.52) U/ gm Hb} and GSH {controls 6.52 +/- 0.25 (95% CI 6.42-6.52), Child B 3.85 +/- 0.18 (95%CI 3.74-3.96), Child C 2.99 +/- 0.30 (95% CI 2.82-3.16) mmol/ gm Hb}. CONCLUSIONS : Oxidative stress is associated with the development and progression of cirrhosis.

Attenuation of phosphamidon-induced oxidative stress and immune dysfunction in rats treated with N-acetylcysteine

The effect of N-acetylcysteine, a thiolic antioxidant, on attenuation of phosphamidon-induced oxidative stress and immune dysfunction was evaluated in adult male Wistar rats weighing 200-250 g. Rats were divided into four groups, 8 animals/group, and treated with phosphamidon, N-acetylcysteine or the combination of both for 28 days. Oral administration of phosphamidon (1.74 mg/kg), an organophosphate insecticide, increased serum malondialdehyde (3.83 ± 0.18 vs 2.91 ± 0.24 nmol/mL; P < 0.05) and decreased erythrocyte superoxide dismutase (567.8 ± 24.36 vs 749.16 ± 102.61 U/gHb; P < 0.05), catalase activity (1.86 ± 0.18 vs 2.43 ± 0.08 U/gHb; P < 0.05) and whole blood glutathione levels (1.25 ± 0.21 vs 2.28 ± 0.08 mg/gHb; P < 0.05) showing phosphamidon-induced oxidative stress. Phosphamidon exposure markedly suppressed humoral immune response as assessed by antibody titer to ovalbumin (4.71 ± 0.51 vs 8.00 ± 0.12 -log2; P < 0.05), and cell-mediated immune response as assessed by leukocyte migration inhibition (25.24 ± 1.04 vs 70.8 ± 1.09%; P < 0.05) and macrophage migration inhibition (20.38 ± 0.99 vs 67.16 ± 5.30%; P < 0.05) response. Phosphamidon exposure decreased IFN-у levels (40.7 ± 3.21 vs 55.84 ± 3.02 pg/mL; P < 0.05) suggesting a profound effect of phosphamidon on cell-mediated immune response. A phosphamidon-induced increase in TNF-α level (64.19 ± 6.0 vs 23.16 ± 4.0 pg/mL; P < 0.05) suggests a contributory role of immunocytes in oxidative stress. Co-administration of N-acetylcysteine (3.5 mmol/kg, orally) with phosphamidon attenuated the adverse effects of phosphamidon. These findings suggest that oral N-acetylcysteine treatment exerts protective effect and attenuates free radical injury and immune dysfunction caused by subchronic phosphamidon exposure.

Melatonin treatment prevents modulation of cell-mediated immune response induced by propoxur in rats.

The effect of melatonin, a major secretory product of the pineal gland, in attenuation of propoxur (2-isopropoxy phenyl N-methyl carbamate)-induced modulation of cell-mediated immune (CMI) response was studied in rats. Male Wistar albino rats were exposed to propoxur (a widely used pesticide) orally (10 mg/kg) and/or melatonin (10 mg/kg) orally for 4 weeks. CMI was measured by delayed-type hypersensitivity (DTH), leucocyte and macrophage migration inhibition (LMI and MMI) responses and estimation of cytokines TNF-alpha and IFN-gamma levels. Rats exposed to propoxur for 4 weeks showed significant decrease in DTH, LMI and MMI responses. Propoxur also suppressed TNF-alpha and IFN-gamma production significantly. Administration of melatonin alone caused a significant increase in DTH response. Although there were no changes in the LMI and MMI response, the cytokine levels were significantly increased, as compared to control. Co-administration of melatonin along with propoxur significantly nullified the effect of the pesticide on the CMI response, except DTH and reversed levels of cytokines to near control/normal values. Thus, melatonin treatment considerably attenuated immunomodulation caused by sub-chronic treatment of propoxur in experimental animals.

Advanced glycosylated end products-mediated activation of polymorphonuclear neutrophils in diabetes mellitus and associated oxidative stress.

Two important consequences of hyperglycemia in diabetes are development of oxidative stress and formation of advanced glycation end products (AGE) which are known to be associated with diabetic complications. Relationship between AGE formation and development of oxidative stress (OS) is yet to be established. In the present study, the involvement of AGE in PMN-mediated ROS generation and the associated OS were investigated in type 2 diabetic mellitus (DM) patients. We assessed OS parameters (serum MDA, FRAP and GSH), PMN oxidative functions (respiratory burst and superoxide production) and total serum AGE in 90 subjects divided equally in three groups--control group, Group I consisting of type 2 diabetic patients without microvascular complications and Group II consisting of type 2 diabetic patients with microvascular complications. PMNs isolated from both groups (I and II) exhibited higher level of respiratory burst (RB) and produced increased amount of superoxide anion as compared to the controls. The increase was more pronounced in diabetes with complications, as compared to those without. Serum malondialdehyde (MDA) level was elevated, whereas glutathione (GSH) and ferric reducing ability of plasma (FRAP) levels were significantly reduced in diabetes as compared to the controls, suggesting the presence of oxidative stress in DM. A positive correlation between PMN oxidative function and OS parameters suggested the involvement of PMN in the development of OS in DM. Serum AGE level was also elevated in diabetic groups as compared to the controls. Further, the positive correlation between serum AGE level and PMN oxidative function suggested the involvement of AGE in increased RB and generation of reactive oxygen species (ROS) by resting diabetic PMN. The results of the study indicate that AGE-PMN interaction possibly upregulates NADPH oxidase, leading to enhanced ROS generation and thus contributes to the pathogenesis in diabetes.

Immunotoxicity of phosphamidon following subchronic exposure in albino rats.

Effect of subchronic doses of phosphamidon exposure on humoral and cell mediated immune (CMI) responses were studied in male albino rats using SRBC, ovalbumin and KLH as antigens. Humoral immune responses were assessed by estimating antibody titre against antigen and splenic plaque forming cells (PFC) assay. CMI responses were studied by using leucocyte migration inhibition (LMI), macrophage migration inhibition (MMI) and delayed type hypersensitivity (DTH) response. Results obtained in the present study revealed marked suppression of humoral and CMI responses in a dose dependent pattern. Hence, suppression of immune responses by phosphamidon even at subchronic doses is clearly an important aspect for its safety evaluation.

Protective effect of melatonin against propoxur-induced oxidative stress and suppression of humoral immune response in rats.

Effect of melatonin in attenuation of propoxur induced oxidative stress and suppression of humoral immune response was studied in rats. Oral administration of propoxur (10 mg/kg) increased lipid peroxidation in serum after 28 days treatment. Superoxide dismutase, catalase and glutathione were also altered following propoxur exposure. In addition propoxur exposure markedly suppressed humoral immune response as assessed by antibody titre and plaque forming cell assay. Simultaneous treatment with melatonin (5 mg/kg, ip) markedly attenuated the effect of propoxur on (a) lipid peroxidation, (b) oxidative stress parameters and (c) immunotoxicity. Results have been discussed in the light of possible immunopotentiating and antioxidant effects of melatonin to understand the influence of oxidative stress on propoxur induced immunomodulation.

Influence of dietary ginger (Zingiber officinales Rosc) on antioxidant defense system in rat: comparison with ascorbic acid.

Ginger (Z. officinale; 1% w/w) significantly lowered lipid peroxidation by maintaining the activities of the antioxidant enzymes--superoxide dismutase, catalase and glutathione peroxidase in rats. The blood glutathione content was significantly increased in ginger fed rats. Similar effects were also observed after natural antioxidant ascorbic acid (100 mg/kg, body wt) treatment. The results indicate that ginger is comparatively as effective as ascorbic acid as an antioxidant.

Organochlorine pesticide-induced oxidative stress and immune suppression in rats.

Effects of subchronic DDT and lindane exposure were evaluated on lipid peroxidation, antioxidant mechanisms and humoral immune response in rats. Oral administration of DDT, (100 and 200 ppm) and lindane (40 and 80 ppm) dose dependently increased thiobarbituric acid reactive substance (TBARS) levels in serum after 8 wk of treatment. Superoxide dismutase (SOD) activity in red blood cells (RBC) was also dose dependently increased by these compounds. In addition, such DDT or lindane exposure markedly suppressed the humoral immune response as assessed by anti-sheep RBC antibody titres. Simultaneous treatment with ascorbic acid (100 mg/kg) markedly attenuated the effects of DDT and lindane on (a) lipid peroxidation, (b) SOD activity and (c) humoral immune suppression. These results indicate the possible involvement of free radicals in organochlorine-induced immunotoxicity.

A comparative evaluation of immunotoxicity of malathion after subchronic exposure in experimental animals.

The effects of sub-chronic doses of malathion exposure on humoral and cell-mediated immune (CMI) responses were studied in male albino mice, rats and rabbits using sheep red blood cells (SRBC), tetanus toxoid and ovalbumin as antigens. The humoral immune response was assessed by estimating serum immunoglobulin (IgM and IgG) concentrations, antibody titre against antigens and splenic-plaque forming cells (PFC). The CMI response was studied by using the leucocyte migration inhibition (LMI) and macrophage migration inhibition (MMI) tests. In general there were (a) attenuation in antigen induced antibody response, (b) suppression of PFC, and (c) marked inhibition of LMI and MMI factors. Sub-chronic malathion exposure induced differential degrees of humoral and CMI suppression in these experimental animals. However, both cellular and humoral immune responses were decreased in a dose-time dependent pattern and a consistent trend was observed. The threshold level of the malathion for inducing immune suppression depends on the animal species, type of antigen used, and the method of immunological assay. In view of the widespread use of malathion a comparative assessment of immune responses using different experimental animals and antigens is an important aspect of its safety evaluation.

Modulation of gamma glutamyl transpeptidase activity in lymphoid system by organochlorine pesticides in rats.

Effects of 1,1,1,-trichloro-2,2-bis (p-chlorophenyl) ethane (DDT) and lindane were studied on gamma glutamyl transpeptidase (GGT) activity in different tissues of the lymphoid system in rats. DDT (100 or 200 ppm) and lindane (30 or 80 ppm) exposure for 8 weeks suppressed the GGT activity in a dose dependent manner in thymus and macrophage. In spleen, significant decrease in the enzyme activity was observed at higher exposure (200 ppm DDT or 80 ppm lindane) levels. Lindane suppressed GGT activity at both 30 or 80 ppm dose levels, while DDT reduced the GGT activity at 200 ppm but not at 100 ppm exposure in lymphocyte. The study indicates the possibility of using GGT as an effective and consistent biochemical marker for immunotoxicity of xenobiotics and other environmental stressors.

Effects of stress on gamma glutamyl transpeptidase (GGT) activity in lymphoid system of rats: modulation by drugs.

Effects of stress and its modulation by adaptogens were evaluated on gamma glutamyl transpeptidase (GGT) activity in different tissues of the lymphoid system in rats. Restrain stress (RSx5) suppressed the GGT activity in different tissues of lymphoid system viz. the lymphocyte, the spleen, the thymus and the macrophage, and the maximum effect was seen in the spleen. Chlordiazepoxide, a prototype anti-stress agent, which did not alter GGT activity per se, reversed the effect of RS on this enzyme activity in tissues of lymphoid system studied. Azardirachta indica (Al, Neem), an indigenous adaptogen stimulated the GGT activity per se and nearly normalised RS induced suppression of GGT in lymphoid system. The observed suppression of GGT activity in lymphoid system by stress and its modulation by natural and synthetic adaptogens indicates that GGT could be a consistent cellular/biochemical marker of stress responsiveness and stress-induced immunomodulation.

Influence of subchronic exposure to lindane on humoral immunity in mice.

Lindane suppressed both primary and secondary antibody responses to sheep red blood cell (SRBC) in albino mice, the effects being more pronounced on the secondary than the primary response. However, a longer duration of pesticide exposure induced similar degrees of immunosuppression on both responses. The sequential study of plaque forming cells (PFC) kinetics revealed that suppression of plaque formation not only occurred at peak days but also on pre and post peak days, and there was no delay in peak antibody formation. Moreover, reduction in the primary PFC was not associated with decrease in the antibody response to SRBC. The results indicate that lindane suppresses both primary and secondary humoral immune responses in a time and dose dependent manner, and suggest a threshold susceptibility to exposure.

Immunotoxicity of pesticides: perspectives and trends.

In recent years, great concern has been expressed about genotoxic potential of pesticide chemicals. These toxic chemicals have become an integral part of the ecosystem and the human health effects of these agents are yet to be satisfactorily defined. The objectives of this review is to examine the sources of information available; evaluation of experimental protocols employed for assessment of immunological effects; and to study specific cellular and molecular locus which could be responsible for impaired immune responsiveness. It is emphasized that threshold level for the pesticide effect below which no effect would be seen, depends on the animal species, the method of testing for immune responses and type of antigen used. A comparative assessment of immune responses using different antigens is, therefore, an important aspect of pesticide immunotoxicity. In view of widespread use, distribution and stability of some of these compounds in the environment, pesticide exposure may play a greater role in suspected fragile immune system, and may result in altered disease susceptibility. An understanding of these risks depends, to a great extent, upon cellular and molecular events underlying pesticide-induced immune alterations in experimental animals. It is, therefore, proposed that pesticide chemicals may influence humoral immunity while having no detectable effect on cell-mediated immunity (CMI); immune dysfunction is related to dose and duration of pesticide exposure; a single assay of immune function may not be appropriate to detect pesticide-induced immune dysfunction; since many immune responses are genetically controlled, alterations in responsiveness to one challenge in a given animal model may not hold true in second one; although it has been established that pesticide chemicals can alter immune function, the mechanisms of action have yet to be determined. This paper also reviews the effects of pesticide on lymphocyte function and suggests that lymphocyte dysfunction may be an integral part of pesticide-induced immunosuppression and presents an approach which may serve to delineate the possible mechanisms of action. It is quite clear that pesticide-induced immunomodulation endangers humans and animals. This hazard should, therefore, not to be underestimated in evaluation of toxicity of these chemicals. However, additional research is needed in basic mechanism of immunotoxicity and identification of susceptibility factors which predispose to these reactions.

Modulation of humoral and cell-mediated immune responses by Azadirachta indica (Neem) in mice.

The effects of A. indica (AI, Neem) were evaluated on tests of humoral and cell-mediated immune responses after 3 weeks of oral AI (leaf extract) treatment in ovalbumin immunized mice. At the dose levels tested, AI (10, 30 or 100 mg/kg), had no appreciable influence on different organ (liver, spleen, thymus)/body weight indices, when compared to controls. In tests for humoral immune responses, AI (100 mg/kg) treated mice had higher (1) IgM and IgG levels, and (b) anti-ovalbumin antibody titres, when compared to the vehicle treated group. In tests for cell-mediated immune responses, there was an enhancement (%) of (a) macrophage migration inhibition, and (b) footpad thickness after AI (100 mg/kg) treatment. These results are discussed in light of the possible immunopotentiating effects of AI.

A comparative evaluation of immunotoxicity of DDT and its metabolites in rats.

Effects of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) and its metabolites, 1,1-dichloro-2,2-bis(p-chlorophenyl)ethene (DDE), 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD) and 2,2-bis(p-chlorophenyl) acetic acid (DDA) were comparatively evaluated on humoral and cell mediated immune (CMI) responses in rats. Rats were given a diet containing 200 ppm of the various test compounds for 6 weeks and were subsequently immunized with ovalbumin. DDT, DDE and DDD, all induced differential degrees of humoral and cellular immune suppression. There were (a) increases in albumin/globulin ratios, (b) suppression of IgM and IgG levels, and (c) attenuations in ovalbumin induced antibody responses. In CMI studies, there were marked inhibitions of (a) leucocyte and macrophage migration factors, and (b) delayed type hypersensitivity (DTH) reaction. Whereas, these effects were most marked with DDE and DDD, DDA did not elicit such immunomodulatory effects. It is inferred that suppression of immune responses by immediate DDT metabolites, DDE (and DDD and not DDA) is an important determinant of the toxicity of DDT (DDE > DDD > DDT) and the influence of this environmental pollutant in health and disease.

Influence of dietary protein on DDT-induced immune responsiveness in rats.

The influence of protein deficiency was evaluated on immune responsiveness after subchronic DDT exposure in albino rats. Rats were given 20%, 12% and 3% protein diets and exposed to DDT (20, 50 or 100 ppm) for 4 weeks. DDT (50 and 100 ppm) induced humoral and cellular immune suppression only in rats fed on 3% protein diet. There was (a) an increase in the albumin/globulin ratio, (b) suppression in IgM and IgG levels, and (c) attenuation in the tetanus toxoid-induced antibody responses. Further, in rats immunized with tetanus toxoid, the leucocyte and macrophage migration inhibition were also attenuated. Moreover, these animals maintained on 3% protein diet showed depression in humoral and cellular immune responses to antigen in a dose-dependent pattern after exposure to DDT at dose levels which were not immunosuppressive for rats on 12% or 20% protein diet. These results suggest that dietary protein content may predispose to the immunotoxic effects of DDT exposure, and also be a crucial determinant in DDT detoxification.