M. F. Rahman

Assessment of genotoxic effects of chloropyriphos and acephate by the comet assay in mice leucocytes.

Two organophosphorus (OP) pesticides (chloropyriphos and acephate) and cyclophosphamide (CP) (positive control) were tested for their ability to induce in vivo genotoxic effect in leucocytes of Swiss albino mice using the single cell gel electrophoresis assay or comet assay. The mice were administered orally with doses ranging from 0.28 to 8.96 mg/kg body weight (b. wt.) of chloropyriphos and 12.25 to 392.00 mg/kg b.wt. of acephate. The assay was performed on whole blood at 24, 48, 72 and 96 h. A significant increase in mean comet tail length indicating DNA damage was observed at 24h post-treatment (P<0.05) with both pesticides in comparison to control. The damage was dose related. The mean comet tail length revealed a clear dose dependent increase. From 48 h post-treatment, a gradual decrease in mean tail length was noted. By 96 h of post-treatment the mean comet tail length reached control levels indicating repair of the damaged DNA. From the study it can be concluded that the comet assay is a sensitive assay for the detection of genotoxicity caused by pesticides.

Genotoxic effect of monocrotophos to sentinel species using comet assay

Monocrotophos is the single largest selling agrochemical in India. Sensitive biomarkers to study the genotoxic effects caused by monocrotophos in aquatic organisms, especially fish, are lacking. The fish used in this study are Tilapia mossambica, which are edible, commercially valuable and distributed all over India. The objective of this study was to study DNA strand breaks induced by monocrotophos in T. mossambica in vivo using single-cell micro gel electrophoresis/comet assay. Tilapia were treated orally with 0.313, 0.625, 1.25, 1.875, 2.5, 3.125, 3.75 and 4.375 ppm of monocrotophos and the assay was performed on nucleated erythrocytes after 24, 48, 72 and 96 h. A significant increase in mean comet tail-length (5.21-7.46 microM), indicating DNA damage, was observed at all the doses with monocrotophos when compared to controls (3.36 microM). The mean tail-length showed a dose-related increase and time-dependent decrease. The maximum increase in mean comet tail-length was observed at 24 h. Relative to these effects, reductions in mean comet tail-length were seen at 48 and 72 h. By 96 h, values had returned to control levels at all doses, indicating repair of the damaged DNA and/or loss of heavily damaged cells. The study reveals that the comet assay is a sensitive and rapid method to detect genotoxicity of monocrotophos and other environmental pollutants in sentinel species.

Effects of Vepacide (Azadirachta indica) on aspartate and alanine aminotransferase profiles in a subchronic study with rats

The aim of this study was to ascertain the long-term effects of Vepacide, a neem-based pesticide on biochemical profiles. Albino Wistar rats were treated orally with 80 (low), 160 (medium) and 320 mg/kg (high) doses of Vepacide in coconut oil for 90 days. Control rats received the same volume of the vehicle. Vepacide caused increase of aspartate and alanine aminotransferase in serum, kidney and lung, and these enzymes decreased in liver in both male and female rats when measured after 45 and 90 days of treatment. The two-way analysis of variance (ANOVA) showed that the alterations in these enzymes were dose–and time-dependent. Sexual dimorphism was observed when male rats were compared with female rats (Student t-test at P< 0.05). Positive correlation was observed with regard to these enzymes between serum, kidney and lung, whereas in the case of serum and liver, a negative correlation was recorded. These enzyme profiles elucidate that they increased in serum with simultaneous decrease in liver, indicating necrosis of liver, whereas in other tissues, the level of enzymes increased, showing an adaptive mechanism due to the chemical stress. The affected enzymes were recovered to normal conditions after 28 days of post-treatment (withdrawal study). Due to the Vepacide treatment, lung was more affected followed by liver and kidney. This study has indicated that these enzymes could be useful as biomarkers for the insult of any toxicant. Besides, they can also help in predictive toxicology.

Genotoxicity evaluation in workers occupationally exposed to lead

Lead (Pb) is a widely used heavy metal with a broad industrial usage. Nevertheless, Pb is a serious public health issue as it is one of the most widespread environmental and industrial toxins. The aim of this investigation was to assess the genotoxicity of Pb using the comet assay, micronucleus (MN) and chromosomal aberrations (CA) test. Blood and urinary Pb content, levels of delta-aminolevulinic acid dehydratase in the erythrocytes (E-ALAD) and delta-aminolevulinic acid in urine (U-ALA) were determined. The exposure associated oxidative stress was also studied. The study group comprised of 90 male Pb recovery unit workers and 90 matched controls. The results indicated that the exposed workers had a significantly higher mean comet tail length than that of controls (P<0.05). Analysis of micronuclei in buccal epithelial cells (BECs) and peripheral blood lymphocytes (PBL) revealed that there was a significant increase in frequency of MN in exposed subjects than controls. The frequency of aberrant metaphases was also found to be significantly elevated in the Pb exposed workers. The levels of antioxidant enzymes were relatively reduced (P>0.05) while the rate of lipid peroxidation was higher in the exposed subjects. Blood and urinary Pb concentrations were found to be higher in exposed workers than in controls. E-ALAD levels were reduced and U-ALA levels were elevated in the exposed subjects in comparison to controls. Results of analysis, taking the confounding factors into consideration provide evidence for the association of Pb exposure and genotoxicity, and predict the increased risk of cancer to the exposed workers. In view of the observed results, it can be strongly concluded that the workers comprise the risk group and adequate safety, precautionary and preventive measures could only minimize exposure and the related health hazards.

In vivo genotoxicity assessment of aluminium oxide nanomaterials in rat peripheral blood cells using the comet assay and micronucleus test

Advances in nanotechnology and its usage in various fields have led to the exposure of humans to engineered nanomaterials (NMs) and there is a need to tackle the potential human health effects before these materials are fully exploited. The main purpose of the current study was to assess whether aluminium oxide NMs (Al(2)O(3)-30 nm and Al(2)O(3)-40 nm) could cause potential genotoxic effects in vivo. Characterization of Al(2)O(3)-30 nm and Al(2)O(3)-40 nm was done with transmission electron microscopy, dynamic light scattering and laser Doppler velocimetry prior to their use in this study. The genotoxicity end points considered in this study were the frequency of micronuclei (MN) and the percentage of tail DNA (% Tail DNA) migration in rat peripheral blood cells using the micronucleus test (MNT) and the comet assay, respectively. Genotoxic effects were evaluated in groups of female Wistar rats (five per group) after single doses of 500, 1000 and 2000 mg/kg body weight (bw) of Al(2)O(3)-30 nm, Al(2)O(3)-40 nm and Al(2)O(3)-bulk. Al(2)O(3)-30 nm and Al(2)O(3)-40 nm showed a statistically significant dose-related increase in % Tail DNA for Al(2)O(3)-30 nm and Al(2)O(3)-40 nm (P < 0.05). However, Al(2)O(3)-bulk did not induce statistically significant changes over control values. The MNT also revealed a statistically significant (P < 0.05) dose-dependent increase in the frequency of MN, whereas Al(2)O(3)-bulk did not show any significant increase in frequency of MN compared to control. Cyclophosphamide (40 mg/kg bw) used as a positive control showed statistically significant (P < 0.001) increase in % Tail DNA and frequency of MN. The biodistribution of Al(2)O(3)-30 nm and Al(2)O(3)-40 nm and Al(2)O(3)-bulk in different rat tissues, urine and feces was also studied 14 days after treatment using inductively coupled plasma mass spectrometry. The data indicated that tissue distribution of Al(2)O(3) was size dependent. Our findings suggest that Al(2)O(3) NMs were able to cause size- and dose-dependent genotoxicity in vivo compared to Al(2)O(3)-bulk and control groups.

ACID AND ALKALINE PHOSPHATASE ACTIVITIES IN A NOVEL PHOSPHOROTHIONATE (RPR-11) TREATED MALE AND FEMALE RATS. EVIDENCE OF DOSE AND TIME-DEPENDENT RESPONSE

The effect of a novel phosphorothionate, the methyl ester of 2-butenoic acid-3-diethoxy phosphinothioyl (RPR-II) was studied on membrane bound target enzymes Acid (AcP) and Alkaline (AkP) Phosphatases in different tissues of male and female albino Wistar rats. Three sub-chronic doses 0.014 (low), 0.028 (medium) and 0.042 (high) mg/kg-1 were administered to the rats daily for a period of 90 days. The long term and repeated administration of RPR-II caused significant increase of AcP and AkP in serum and kidney (AcP), whereas these enzymes simultaneously decreased significantly in liver, kidney (female rat AkP) and lung tissues in both male and female rats after 45 and 90 days of treatment. However, the kidney AcP increased significantly in both the sexes which is suggestive of an increase in synthesis of this enzyme which may be an adaptive mechanism to the toxicant stress. The changes in serum, liver, kidney and lung of both male and female rats by this compound were statistically significant when compared with two way Anova showing that they are dose and time dependent. The alterations in male rats were statistically insignificant when compared with female rats showing no sexual dimorphism by this compound. Recovery was observed after 28 days of post treatment (withdrawal study) indicating reversal of the toxic symptoms once the toxicant is removed. High degree negative correlation was observed for serum versus liver and lung and in other cases substantial correlation was observed. The changes observed in these enzymes showed that liver was most susceptible followed by lung and kidney. There are marker enzymes and their increase in different tissues might be due to the increased permeability of plasma membrane or cellular necrosis, showing the stress condition of the treated rats. This investigation elucidates the effect of these biomarker enzymes which increased in blood, might be due to the necrosis of liver, kidney and lung tissues by this compound.

Comparative study of genotoxicity and tissue distribution of nano and micron sized iron oxide in rats after acute oral treatment.

Though nanomaterials (NMs) are being utilized worldwide, increasing use of NMs have raised concerns over their safety to human health and environment. Iron oxide (Fe(2)O(3)) NMs have important applications. The aim of this study was to assess the genotoxicity of Fe(2)O(3)-30nm and Fe(2)O(3)-bulk in female Wistar rats. Fe(2)O(3)-30nm was characterized by using transmission electron microscopy, dynamic light scattering, laser Doppler velocimetry and surface area analysis. The rats were treated orally with the single doses of 500, 1000, 2000mg/kg bw of Fe(2)O(3)-30nm and Fe(2)O(3) -bulk. The genotoxicity was evaluated at 6, 24, 48 and 72h by the comet assay in leucocytes, 48 and 72h by micronucleus test (MNT) in peripheral blood cells, 18 and 24h by chromosomal aberration (CA) assay and 24 and 48h by MNT in bone marrow cells. The biodistribution of iron (Fe) was carried out at 6, 24, 48 and 72h after treatment in liver, spleen, kidney, heart, brain, bone marrow, urine and feces by using atomic absorption spectrophotometry. The % tail DNA, frequencies of micronuclei and CAs were statistically insignificant (p>0.05) at all doses. These results suggest that Fe(2)O(3)-30nm and Fe(2)O(3)-bulk was not genotoxic at the doses tested. Bioavailability of Fe was size and dose dependent in all the tissues from the groups exposed to Fe(2)O(3)-30nm. Fe(2)O(3) NMs were able to enter in the organs and the rats are biocompatible with much higher concentration of Fe. However, the accumulated Fe did not cause significant genotoxicity. This study provides additional knowledge about the toxicology of Fe(2)O(3) NMs.

Evaluation of genotoxic effects of oral exposure to aluminum oxide nanomaterials in rat bone marrow.

Nanomaterials have novel properties and functions because of their small size. The unique nature of nanomaterials may be associated with potentially toxic effects. The aim of this study was to evaluate the in vivo genotoxicity of rats exposed with Aluminum oxide nanomaterials. Hence in the present study, the genotoxicity of Aluminum oxide nanomaterials (30 and 40 nm) and its bulk material was studied in bone marrow of female Wistar rats using chromosomal aberration and micronucleus assays. The rats were administered orally with the doses of 500, 1000 and 2000 mg/kg bw. Statistically significant genotoxicity was observed with Aluminum oxide 30 and 40 nm with micronucleus as well as chromosomal aberration assays. Significantly (p < 0.05 or p < 0.001) increased frequency of MN was observed with 1000 and 2000 mg/kg bw dose levels of Aluminum oxide 30 nm (9.4 +/- 1.87 and 15.2 +/- 2.3, respectively) and Aluminum oxide 40 nm (8.1 +/- 1.8 and 13.9 +/- 2.21, respectively) over control (2.5 +/- 0.7) at 30 h. Likewise, at 48 h sampling time a significant (p < 0.05 or p < 0.001) increase in frequency of MN was evident at 1000 and 2000 mg/kg bw dose levels of Aluminum oxide 30 nm (10.6 +/- 1.68 and 16.6 +/- 2.66, respectively) and Aluminum oxide 40 nm (9.0 +/- 1.38 and 14.7 +/- 1.68, respectively) compared to control (1.8 +/- 0.75). Significantly increased frequencies (p < 0.05 or p < 0.001) of chromosomal aberrations were observed with Aluminum oxide 30 nm (1000 and 2000 mg/kg bw) and Aluminum oxide 40 nm (2000 mg/kg bw) in comparison to control at 18 and 24 h. Further, since there is need for information on the toxicokinetics of nanomaterials, determination of these properties of the nanomaterials was carried out in different tissues, urine and feces using inductively coupled plasma mass spectrometry (ICP-MS). A significant size dependent accumulation of Aluminum oxide nanomaterials occurred in different tissues, urine and feces of rats as shown by ICP-MS data. The results of our study suggest that exposure to Aluminum oxide nanomaterials has the potential to cause genetic damage.

Toxicity Study of Cerium Oxide Nanoparticles in Human Neuroblastoma Cells

The present study consisted of cytotoxic, genotoxic, and oxidative stress responses of human neuroblastoma cell line (IMR32) following exposure to different doses of cerium oxide nanoparticles (CeO2 NPs; nanoceria) and its microparticles (MPs) for 24 hours. Cytotoxicity was evaluated by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and lactate dehydrogenase assays whereas genotoxicity was assessed using the cytokinesis-block micronucleus and comet assays. A battery of assays including lipid peroxidation, reactive oxygen species (ROS), hydrogen peroxide, reduced glutathione, nitric oxide, glutathione reductase, glutathione peroxidase, superoxide dismutase, catalase, and glutathione S-transferase were performed to test the hypothesis that ROS was responsible for the toxicity of nanoceria. The results showed that nanosized CeO2 was more toxic than cerium oxide MPs. Hence, further study on safety evaluation of CeO2 NPs on other models is recommended.

In vitro mutagenicity assessment of aluminium oxide nanomaterials using the Salmonella/microsome assay

The aim of the current study was to evaluate the potential mutagenicity of aluminium oxide nanomaterials (NMs) (Al(2)O(3)-30 nm and Al(2)O(3)-40 nm). Characterization of the NMs was done before the initiation of the study. The mutagenicity of the NMs was studied by the Ames test with Salmonella typhimurium TA100, TA1535, TA98, TA97a and TA102 strains, in the presence and absence of the S9 mixture. Based on a preliminary cytotoxicity study conducted on the strains, different concentrations of Al(2)O(3)-30 nm, Al(2)O(3)-40 nm and Al(2)O(3)-bulk were selected. At all the concentrations tested, Al(2)O(3)-30 nm and Al(2)O(3)-40 nm did not significantly increase the number of revertant colonies compared to the Al(2)O(3)-bulk and control with or without S9 mixture. Our findings suggest that Al(2)O(3) NMs were devoid of any size and concentration dependent mutagenicity compared to the Al(2)O(3)-bulk and control.