CYTOTOXICITY OF HEXAVALENT CHROMATE COMPOUNDS IN CH310T1/2 CELLS AND CYTOMODULATION BY SODIUM ARSENITE AND METHANOL EXTRACT OF Rauvolfia vomitora (Afzel) IN MICE

Abstract

Exposure to certain hexavalent chromate compounds (HCC) causes lung and colon cancers. Their mechanisms of cytotoxicity are unclear, but believed to be affected by ascorbate and particle size. However, their role is not clearly defined. Co-exposure with sodium arsenite (SA) is common, but its effect on HCC toxicity is unknown. Current therapy has side effects, necessitating the search for antidote from unexplored natural products such as Rauvolfia vomitora (RV). This study therefore investigates the effect of particle size and ascorbate on cytotoxity of selected HCC [lead chromate (PbCrO₄), barium chromate (BaCrO₄), strontium chromate (SrCrO₄) and potassium dichromate (K₂Cr₂O₇)] in C3H10T½ cells and cytomodulatory effects of SA and RV in mice. The effect of ascorbate, dehydroascorbate and panicle size on HCC cytotoxicity in C3H10T½ cells was determined by measuring survival fraction and yield of foci by microscopy. Actin and cellular ultrastructure disruption and induction of cell death were assessed by electron and fluorescent microscopy. The molecular mechanisms of cytotoxicity and transformation were evaluated in eighty-four cell death genes using real time (RT²) gene array, while cell cycle analysis was done by flow cytometry. Leaves of RV were air dried, powdered and extracted with methanol. Forty male mice (20-25g) were divided into 8 groups of 5 Swiss albino mice each and treated with water (control), RV (275 mg/Kg), SA (2.5 mg/kg), K₂Cr₂O₇ (12 mg/Kg), SA + K₂Cr₂O₇, RV + SA, RV + K₂Cr₂O₇, RV + SA + K₂Cr₂O₇. Rauvolfia vomitora was given orally for seven days, while K₂Cr₂O₇ and SA were administered on day seven. Serum aspartate and alanine aminotransferases (AST and ALT), catalase, glutathione-S-transferase (GST), glutathione and malondialdehyde (MDA) levels were determined by spectrophotometry. Micronucleated polychromatic erythrocytes (mPCEs) were evaluated by microscopy. Data were analysed using ANOVA and Student's t- test at p= 0.05. Survival fraction of control cells was 1.0, treatment with PbCrO₄ and ≤12.5µM ascorbate or ≤ 2µM dehydroaseorbate decreased it to 0.4. The 15-20µM ascorbate and 3-4µM dehydroascorbate reversed it to 0.7. Exposure of cells to small (≤3µm) and large particles (≤8µm) of PbCrO₄, BaCrO₄ and SrCr0₄ resulted in a dose-dependent decrease in survival. The total foci were higher for PbCr0₄ (3.8) with large particles and BaCr0₄ (6.6) with small particles Phagocytosis of particles was time-dependent. The HCC treatment led to G2/M and S phase arrest, anucleation, actin disruption and mixed cell death. Thirty-four cell death genes including Bax and Casp3 were up-regulated by 4 folds and six including Bel-2 and Traf2 were down-regulated in treated cells. Twenty-one anti-apoptotic and autophagy genes including Atg5 and Bel-2 were up-regulated in PbCrO₄ transformed cells. The K₂Cr₂O₇ and/or SA significantly increased mPCEs, AST, ALT, catalase and MDA levels while glutathione and GST were reduced. The RV restored the markers towards normal values. Cytotoxicity of chromate compounds is particle size and ascorbate dependent. The cytotoxicity might be due to actin disruption, micronuclei induction and cell cycle arrest. Methanol extract of Rauvolfia vomitora modulated the toxicity in mice.