M. Mahboob

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.

Effect of Organophosphorus and Organochlorine Pesticides (Monochrotophos, Chlorpyriphos, Dimethoate, and Endosulfan) on Human Lymphocytes In‐Vitro

The toxicological profile of the four pesticides described herein characterizes its effects on lymphocytes from peripheral blood from healthy donors. The exposure to all pesticides was by direct interaction/incubation with varying concentrations of the pesticide with blood sample in‐vitro. The dose response relationship in each case was calculated by applying log tables as LC50 values. Cytotoxicity of these pesticides on lymphocytes was measured using the trypan blue dye exclusion technique. Based on LC50 value, all the four pesticides were found to be highly toxic to lymphocyte culture, among them, monocrotophos and endosulfan were the most toxic and dimethoate was the least toxic. The genotoxicity of the pesticides was also determined by comet assay. The results revealed that the pesticides caused increase in the tail length indicating DNA damage. This study suggests that these pesticides have the capacity to alter the genetic material particularly chromosomes in mammalian cultures. The comet assay used in this study was found to be a sensitive and rapid method to detect genotoxicity of pesticide compounds.

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.

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.

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.

In vivo genotoxic effect of zinc sulfate in mouse peripheral blood leukocytes using comet assay

Single stranded DNA breaks induced by Zinc sulfate in mice has been studied in vivo using Alkaline Single Cell Gel Electrophoresis (Comet assay). Mice were administered orally with doses of 5.70, 8.55, 11.40, 14.25, 17.10 and 19.95 mg/kg body weight of zinc sulfate respectively. The samples of whole blood were collected at 24, 48, 72, 96 hr and first week post-treatment and the assay was carried out to determine single strand DNA breaks as represented by comet tail-lengths. Results indicated a significant DNA damage at all the doses after treatment with zinc sulfate when compared to controls showing a clear dose-dependent response (p < 0.05). A gradual decrease in the tail-lengths from 48 hr post-treatment onwards was observed indicating a time dependent decrease in the DNA damage. The study confirms that zinc sulfate causes significant DNA damage at the doses used as revealed by comet assay.

Toxicity assessment of manganese oxide micro and nanoparticles in Wistar rats after 28 days of repeated oral exposure

In the near future, nanotechnology is envisaged for large‐scale use. Hence health and safety issues of nanoparticles (NPs) should be promptly addressed. Twenty‐eight‐day oral toxicity, genotoxicity, biochemical alterations, histopathological changes and tissue distribution of nano and microparticles (MPs) of manganese oxide (MnO2) in Wistar rats was studied. Genotoxicity was assessed using comet, micronucleus and chromosomal aberration assays. The results demonstrated a significant increase in DNA damage in leukocytes, micronuclei and chromosomal aberrations in bone marrow cells after exposure of MnO2‐NPs at 1000, 300 mg kg–1 bw per day and MnO2‐MPs at the dose of 1000 mg kg–1 bw per day. Our findings showed acetylcholinestrase inhibition at 1000 as well as at 300 mg kg–1 bw per day in blood and with all the doses in the brain indicating the toxicity of MnO2‐NPs. Further, the doses significantly inhibited different ATPases in the brain P2 fraction. Significant changes were observed in aspartate aminotransferase (AST), alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) in the liver, kidney and serum in a dose‐dependent manner. MnO2‐MPs at 1000 mg kg–1 bw per day were found to induce significant alterations in biochemical enzymes. A significant distribution was found in all the tissues in a dose‐dependent manner. MnO2‐NPs showed a much higher absorptivity and tissue distribution as compared with MnO2‐MPs. A large fraction of MnO2‐NPs and MnO2‐MPs was cleared by urine and feces. Histopathological analysis revealed that MnO2‐NPs caused alterations in liver, spleen, kidney and brain. The MnO2‐NPs induced toxicity at lower doses compared with MnO2‐MPs. Further, this study did not display gender differences after exposure to MnO2‐NPs and MnO2‐MPs. Therefore, the results suggested that prolonged exposure to MnO2 has the potential to cause genetic damage, biochemical alterations and histological changes. Copyright

EFFECTS OF PHOSPHOROTHIONATE ON THE REPRODUCTIVE SYSTEM OF MALE RATS

Acute and Sub-acute toxic effects of a new pesticide phosphorothionate coded as RPR-V on testis of albino rat were studied. For the acute study, rats received a single dose of 30 mg/kg of RPR-V and sacrificed after 24 hours. For the Subacute study, 1.42 mg/kg/day was administered orally to rats for 10 days and 21 days. Acute exposure of rats to RPR-V brought no change either in the Gonadosomatic Index (GSI) or in the structure of testis or in the serum levels of Testosterone. Similarly, no significant change was observed in the Glutathione S-transferase (GST) activity. But, in testis there was significant increased in the reduced Glutathione (GSH) and Acid Phosphatase (AcP), whereas Alkaline Phosphatase (AkP) levels decreased significantly at 24hr post treatment. On 7thday (withdrawal period) after the cessation of the treatment the GSH, AcP, and AkP levels reached to near control. The sub-acute study revealed a significant post treatment. Due to RPR-V treatment the testis AcP levels increased significantly at 21stday of medication but AkP levels decreased both at 10thand 21stday of post treatment. Histopathological studies showed that after 10thday testis showed considerable loss of spermatozoids and at 21stday complete derangement of cellular organization was observed. Testosterone levels decreased significantly after 10thday and remained significantly low at 21stday. However, withdrawal studies showed a recovery in testis of rat treated with RPR-V. GST, GSH, GSI, AcP and AkP values were recovered, testosterone levels were also recovered but recovery in testis structure remained at a low profile.