A comparison of the in vitro genotoxicity of tri- and hexavalent chromium

Chromium can be found in the environment in two main valence states: hexavalent (Cr(VI)) and trivalent (Cr(III)). Cr(VI) salts are well known human carcinogens, but the results from in vitro studies are often conflicting. Cr(VI) primarily enters the cells and undergoes metabolic reduction; however, the ultimate product of this reduction, Cr(III) predominates within the cell. In the present work, we compared the effects of tri- and hexavalent chromium on the DNA damage and repair in human lymphocytes using the alkaline single cell gel electrophoresis (comet assay). Potassium dichromate induced DNA damage in the lymphocytes, measured as the increase in comet tail moment. The effect was dose-dependent. Treated cells were able to recover within a 120-min incubation. Cr(III) caused greater DNA migration than Cr(VI). The lymphocytes did not show measurable DNA repair. Vitamin C at 50 microM reduced the extent of DNA migration. This was either due to a decrease in DNA strand breaks and/or alkali labile sites induced by Cr(VI) or to the formation of DNA crosslinks by Cr(VI) in the presence of vitamin C. Vitamin C, however, did not modify the effects of Cr(III). Catalase, an enzyme inactivating hydrogen peroxide, decreased the extent of DNA damage induced by Cr(VI) but not the one induced by Cr(III). Lymphocytes exposed to Cr(VI) and treated with endonuclease III, which recognizes oxidized pyrimidines, displayed greater extent of DNA damage than those not treated with the enzyme. Such an effect was not observed when Cr(III) was tested. The results obtained suggest that reactive oxygen species and hydrogen peroxide may be involved in the formation of DNA lesions by hexavalent chromium. The comet assay did not indicate the involvement of oxidative mechanisms in the DNA-damaging activity of trivalent chromium and we speculate that its binding to cellular ligands may play a role in its genotoxicity.     

Oxidation of inactive trivalent chromium to the mutagenic hexavalent form.

Soluble trivalent chromium compounds (chromium potassium sulfate, chromium nitrate, chromium chloride, neochromium and chromium alum) were inactive for Salmonella typhimurium TA100, even at milligram amounts per plate. No effect could be detected either in the absence or in the presence of rat-liver, lung or muscle microsomal fractions, of rat-muscle mitochondria (with or without ATP), of oxidized glutathione (GSSG), or of human serum, plasma or erythrocyte lysates. Conversely, addition of a strongly oxidizing agent (potassium permanganate) resulted in toxic effects in plates incorporating more than 40--80 microgram of compounds and elicited a dose-effect mutagenic response at 10--40 microgram per plate. These effects could be ascribed to oxidation of chromium from the trivalent to the active hexavalent state. Insoluble chromite, as tested in the spot test, was spontaneously mutagenic, owing to contamination of the industrial product with hexavalent chromium. The results obtained may be useful to interpret the findings of carcinogenicity tests and to predict health hazards linked to chromium.     

Comparative studies of chromosomal aberration and mutagenicity of trivalent and hexavalent chromium

The comparative cytogenetic and mutagenic effects between trivalent and hexavalent chromium were investigated. Five chromium compounds, K₂Cr₂O₇ and K₂CrO₄ containing Cr⁶⁺, and Cr(CH₃COO)₃, Cr(NO₃)₃ and CrCl₃ containing Cr³⁺, were examined for their ability to induce chromosomal damage in cultures of human leukocytes, for their reactivity with DNA by a rec-assay system and for mutagenicity in the E. coli Hs30R test system.Chromosome-breaking activity was significantly higher for the compounds with hexavalent than trivalent chromium, the efficiency being in the decreasing order K₂Cr₂O₇ > K₂CrO₄ >> Cr(CHCOO)₃ > Cr(NO₃)₃, CrCl₃. In the rec-assay and mutation assay, hexavalent (K₂Cr₂O₇ and K₂CrO₄) and trivalent Cr(CH₃COO)₃) compounds gave positive results, their mutagenic potential being higher in the same order of clastogenic magnitude.     

Distribution, transformation and bioavailability of trivalent and hexavalent chromium in contaminated soil

The purpose of this study was to investigate solid-phase distribution, transformation, and bioavailability of Cr in Cr(III) and Cr(VI) contaminated soils. The effects of EDTA treatment on solid-phase distribution of Cr in soils were also examined. The results show that Cr in both initially Cr(III)- and Cr(VI)-contaminated soils was mainly present in the organic matter bound fraction. Chromium had similar solid-phase distribution and similar overall binding intensity in both Cr(III)- and Cr(VI)-contaminated soils after a growing season. Transformation between Cr(III) and Cr(VI) took place in both Cr(III)- and Cr(VI)-treated soils. Chromium in the Cr(III)-contaminated soils was mostly present as Cr(III), while Cr in Cr(VI)-treated soils was mainly transformed into Cr(III). About 2% of Cr in native non-treated soils was found as Cr(VI). EDTA treatment increased Cr in soluble and exchangeable fraction in Cr(III)-treated soils. In both Cr(III)- and Cr(VI)-contaminated soils, Cr in oxide bound and organic matter bound     

The cytotoxicity and genotoxicity of particulate and soluble hexavalent chromium in human lung cells

Hexavalent chromium (Cr(VI)) is a human lung carcinogen. Cr(VI) is a particularly important and dangerous carcinogen, because there is widespread exposure to it both occupationally and to the general public. However, despite the potential for widespread exposure and the fact that the lung is its target organ, there are few reports of the genotoxicity of Cr(VI) in human lung cells. Clearly, in order to better understand this carcinogen, its effects in its target cells need to be evaluated. Accordingly, we determined the cytotoxicity and clastogenicity of both particulate (water-insoluble) and soluble Cr(VI) in primary human bronchial fibroblasts (PHBFs). We used lead chromate (PbCrO(4)) and sodium chromate (Na(2)CrO(4)) as prototypical particulate and soluble Cr(VI) salts, respectively. Both compounds induced concentration-dependent cytotoxicity after a 24h exposure in PHBFs. The relative survival was 87, 46, 26 and 2% after exposure to 0.1, 0.5, 1 and 5 microg/cm(2) PbCrO(4), respectively, and 74, 57, 13 and 0% after exposure to 1, 2.5, 5 and 10 microM Na(2)CrO(4), respectively. Similarly, the amount of chromosome damage increased with concentration after 24h exposure to both compounds. Specifically, 0.1, 0.5 and 1 microg/cm(2) PbCrO(4) damaged 15, 34 and 42% of metaphase cells with the total amount of damage reaching 18, 40 and 66 aberrations per 100 metaphases, respectively. PbCrO(4) (5 microg/cm(2)) induced such profound cell cycle delay that no metaphases were found. Na(2)CrO(4) (1 and 2.5 microM) damaged 18 and 33% of metaphase cells with the total amount of damage reaching 19 and 43 aberrations per 100 metaphases, respectively. Na(2)CrO(4) (5 and 10 microM) induced such profound cell cycle delay that no metaphases were found. Overall the data clearly indicate that Cr(VI) compounds are cytotoxic and genotoxic to human lung cells.     

Changes in biomass and photosynthetic parameters of tomato plants exposed to trivalent and hexavalent chromium

Tomato plants were treated for two weeks with different concentrations of Cr(III) or Cr(VI) compounds to compare their toxic effects. The concentration of total Cr in plant tissues increased linearly with its concentration in the growth medium and Cr accumulated largely in the roots, regardless of the form in which it was supplied to the plant. All measured plant growth parameters were negatively affected by Cr, but Cr(VI) showed much more pronounced toxic effects. Leaf net photosynthetic rate (P_N) was decreased by both Cr forms, and the decrease was also greater for Cr(VI). Cr(III) caused no significant effect on leaf stomatal conductance, whereas Cr(VI) reduced it. Cr(VI) also markedly reduced the variable to maximum chlorophyll a fluorescence ratio, measured in dark-adapted leaves.     

Biosorption of trivalent and hexavalent chromium from aqueous systems using shelled Moringa oleifera seeds

Abstract

The present study explores the sorption properties of shelled Moringa oleifera seeds (SMOS) for removal of two environmentally important oxidation states of chromium (trivalent and hexavalent) from an aqueous system on the laboratory scale. Sorption studies reveal the optimum conditions for the removal of 81.02%; Cr (III) and 88.15% Cr (VI) as follows: biomass dosage (4.0 g), metal concentration [25mg/L for Cr (III); 50mg/L for Cr (VI)], contact time (40 minutes) at pH 6.5 and 2.5 respectively. The adsorption data were found to fit well both the Freundlich and Langmuir isotherms. Characterization of the seed powder by FTIR showed the clear presence of amino acid moieties having both positively charged amino and negatively charged carboxylic groups and confirmed that biosorption involves amino acid-chromium interactions. SEM studies of native and exhausted [Cr(III) and Cr(VI)] treated SMOS revealed large spherical clusters having a pore area of 8.66 µm² in the case of native SMOS while dense agglomerated etched dendrite type morphology have a pore area of 0.80 µm² in Cr (III) and 0.78 µm² in Cr (VI) treated SMOS The spent biosorbent was regenerated and found to be effectively reusable for four cycles.     

Comparative toxicity of trivalent and hexavalent chromium compounds in fig moth Ephestia cautella (Walker)

Of the trivalent (CrSO4) and hexavalent (K2Cr2O7) chromium compounds, only the hexavalent produced significant deleterious effects on development and fertility of E. cautella, when eggs were reared on laboratory medium supplemented with different concentrations of these salts.     

Hexavalent chromium-reducing plant growth-promoting rhizobacteria are utilized to bio-fortify trivalent chromium in fenugreek by promoting plant development and decreasing the toxicity of hexavalent chromium in the soil

BackgroundFenugreek is known to have good anti-diabetes properties. Moreover, several studies accounted that the trivalent form of chromium [Cr(III)] also have anti-diabetic properties. However, its hexavalent form i.e., Cr(VI) is known to be highly toxic and carcinogenic to living beings and retarded plant growth even if it is present in low concentration in soil. Many plant growth-promoting rhizobacteria (PGPR) are reported to have the potential to reduce the Cr(VI) into Cr(III) in soil. In view of the above, the present objective was designed to effectively utilize Cr(VI) reducing PGPRs for the growth and development of fenugreek plant in Cr(VI) amended soil, apart from reducing Cr(VI) in soil and fortification of Cr(III) in the aerial part of plants.MethodsThe experiment was carried out to evaluate the effect of Cr(VI)-reducing PGPRs viz. Bacillus cereus (SUCR44); Microbacterium sp. (SUCR140); Bacillus thuringiensis (SUCR186) and B. subtilis (SUCR188) on growth, uptake and translocation of Cr as well as other physiological parameters in fenugreek grown under artificially Cr(VI) amended soil (100 mg kg⁻¹ of Cr(VI) in soil).ResultsThe aforementioned concentration of Cr(VI) in soil cause severe reduction in root length (41 %), plant height (43 %), dry root (38 %) and herb biomass (48 %), when compared with control negative (CN; uninoculated plant not grown in Cr(VI) contaminated soil). However, the presence of Microbacterium sp.˗SURC140 (MB) mitigates the Cr toxicity resulting in improved root length (92 %), plant height (86 %), dry root (74 %) and herb biomass (99 %) as compared with control positive (CP; uninoculated plants grown in Cr(VI) contaminated soil). The maximum reduction in bioavailability (82 %) of Cr(VI) in soil and its uptake (50 %) by the plant were also observed in MB-treated plants. However, All Cr(VI)-reducing PGPRs failed to decrease the translocation of Cr to the aerial parts. Moreover, the plant treated with MB observed diminution in relative water content (13 %), electrolyte leakage (16%) and lipid peroxidation (38 %) as well as higher chlorophyll (37 %) carotenoids (17 %) contents and antioxidants (18%) potential.ConclusionThis study demonstrates that MB can lower the Cr(VI) toxicity to the plant by reducing the bioavailable Cr(VI), consequently reducing the Cr(VI) toxicity level in soil and helping in improving the growth and yield of fenugreek. Additionally, Cr(III) uptakes and translocation may improve the effectiveness of fenugreek in treating diabetes.     

Evaluation of genotoxicity of the indenoisoquinoline analogues of fagaronine and nitidine in Drosophila melanogaster

The wing spot test of Drosophila melanogaster was done to evaluate the genotoxicity of the antitumor indenoisoquinoline analogues of nitidine chloride and fagaronien chloride in larvae. Both compounds have toxic effects but no statistically significant increase in the frequency of spots was detected with the analogue of nitidine chloride. This strongly suggests that this compounds is not mutagenic to Drosophila larvae at the concentrations tested. Results with the analogue of fagaronine chloride were ambiguous. Low mutagenicity was detected in only one of two experiments and in the pooled results at 2 mM but not at 5 mM or 10 mM. These results suggest at best a very weak genotoxic effect but its biological significance needs confirmation by results from other assays.     

Effects of the trivalent and hexavalent chromium on the physicochemical properties of mammalian cell nucleic acids and synthetic polynucleotides

The effects of trivalent (chromium chloride) and hexavalent (potassium dichromate) chromium have been studied on the nucleic acids of cultured mammalian cells (BHK hamster fibroblast line), commercial DNA and RNA, and synthetic polynucleotides of known base composition. Modifications of UV absorption spectra and alterations of thermal denaturation and renaturation patterns have been observed by directly treating purified nucleic acids, as well as by examining nucleic acids extracted from cells treated with chromium compounds.Cr(III) interacts with nucleic acid bases, mostly guanine and cytosine, but also with phosphate groups, leading to deprotonation of bases as well as intramolecular cross-links, sandwich complexes between bases and chelation between bases and phosphates. Such interactions destabilize the DNA structure. On the contrary, stabilization of RNA, due to intramolecular metal bonds between nitrogen bases in GC-rich regions, is mainly produced. The kind of interaction of Cr(III) with nucleic acids is not significantly different when intact BHK cells are treated.Cr(VI) interacts similarly with DNA and RNA giving instead different effects when purified nucleic acids or intact cells are treated. Treatment of purified DNA produces breakages in the polynucleotide chain due to the oxidizing power of Cr(VI). In intact cell treatments, changes in the properties of DNA are observed. These could result from the combined action of Cr(III), produced by the intracellular reduction of Cr(VI) and the oxidizing activity of residual Cr(VI).The relevance of Cr(VI) and Cr(III) interactions to the mechanisms of chromium (carcino)genic action is summarized. It is stressed that Cr(VI), if not completely reduced to Cr(III) by extracellular and endoplasmic constituents, can reach the cell nucleus and directly interact with DNA.     

The cytotoxicity and genotoxicity of hexavalent chromium in Steller sea lion lung fibroblasts compared to human lung fibroblasts

In this study we directly compared soluble and particulate chromate cytotoxicity and genotoxicity in human (Homo sapiens) and sea lion (Eumetopias jubatus) lung fibroblasts. Our results show that hexavalent chromium induces increased cell death and chromosome damage in both human and sea lion cells with increasing intracellular chromium ion levels. The data further indicate that both sodium chromate and lead chromate are less cytotoxic and genotoxic to sea lion cells than human cells, based on an administered dose. Differences in chromium ion uptake explained some but not all of the reduced amounts of sodium chromate-induced cell death. By contrast, uptake differences could explain the differences in sodium chromate-induced chromosome damage and particulate chromate-induced toxicity. Altogether they indicate that while hexavalent chromium induces similar toxic effects in sea lion and human cells, there are different mechanisms underlying the toxic outcomes.     

The genotoxicity of the anti-cancer drug mitoxantrone in somatic and germ cells of Drosophila melanogaster.

The novel antineoplastic drug mitoxantrone was studied for its genotoxic effects in Drosophila melanogaster. In male germ cells, the clinical preparation Novantrone, the dihydrochloride salt of mitoxantrone, did not induce sex-linked recessive lethal mutations in feeding and injection experiments with adult flies, although statistically the results were inconclusive rather than truly negative. However, the free base mitoxantrone was weakly, but significantly genotoxic in this test (0.14% lethals/mM exposure concentration); this is most probably the result of prolonged exposure. On the other hand, both forms of mitoxantrone assayed were clearly genotoxic in the somatic mutation and recombination test of the wing. This test assays the cells of the proliferating imaginal wing discs of larvae. Depending on the feeding method used, the overall clone induction frequency was in the range of about 2-6 x 10(-5) per cell and cell generation and per mM exposure dose. Correction of these frequencies according to mean clone size led to slightly higher estimates (by about 5-25% higher). Although the majority of the clone induction events are due to mitotic recombination, a significant proportion can be attributed to mutational events (gene and chromosome mutations). The genotoxicity of mitoxantrone seems to depend mainly on impaired DNA synthesis in cycling cells owing to the compound's ability to inhibit topoisomerase II by intercalation into DNA.     

The genotoxicity of chromium(VI) oxide in the wing spot test of Drosophila melanogaster is over 90% due to mitotic recombination.

Chromium(VI) oxide and chromium(III) chloride were tested in the wing somatic mutation and recombination test (SMART) in Drosophila melanogaster according to standard procedures. The hexavalent compound was highly genotoxic in both chronic and acute treatments whereas the trivalent one was clearly negative. Further analysis of wings carrying an inversion chromosome which eliminates all recombination events showed that over 90% of the spots induced by chromium(VI) oxide are due to mitotic recombination.     

Cytogenetic effects of hexavalent chromium in Bulgarian chromium platers

The aim of the present study was to evaluate the genotoxic effects of hexavalent chromium (Cr(VI)) in vivo in exposed Bulgarian chromium platers by using classical cytogenetic and molecular cytogenetic analyses of peripheral lymphocytes and exfoliated buccal cells. No significant difference was observed between the exposed workers and the controls with regard to the frequency of cells with chromosome aberrations (CAs) using conventional Giemsa staining and in the frequency of sister chromatid exchanges (SCEs). However, there was a significant increase in the number of cells with micronuclei (MN) in peripheral lymphocytes from chromium exposed workers as compared to the controls. In the buccal cells from these workers, this increase was even more pronounced. Cytosine arabinoside (AraC), an inhibitor of DNA synthesis and repair, was found to significantly increase the levels of MN in vitro in the lymphocytes of both groups. The increase was more expressed in the lymphocytes of chromium exposed workers. Both centromere positive (C(+)) as well as centromere negative (C(-)) MN were observed by the fluorescence in situ hybridization (FISH) technique in both of the cell types studied. No difference between C(+) and C(-) MN frequencies was found in the lymphocytes as well as in the buccal cells. Thus, Cr(VI) appears to have both clastogenic as well as aneugenic effects in humans.     

Bacterial reduction of hexavalent chromium

Summary Cr(VI)-reducing bacteria are widespread and Cr(VI) reduction occurs under both aerobic and anaerobic conditions. Under aerobic conditions, both NADH and endogenous cell reserves may serve as the electron donor for Cr(VI) reduction. Under anaerobic conditions, electron transport systems containing cytochromes appear to be involved in Cr(VI) reduction. High cell densities are necessary to obtain a significant rate of Cr(VI) reduction. Cr(VI) reduction by bacteria may be inhibited by Cr(VI), oxygen, heavy metals, and phenolic compounds. The optimum pH and temperature observed for Cr(VI) reduction generally coincide with the optimal growth conditions of cells. The optimum redox potential for Cr(VI) reduction has not yet been established.     

Comparative toxicity of trivalent and hexavalent chromium to rabbits. III. Biochemical and histological changes in testicular tissue.

The administration of trivalent and hexavalent chromium compounds produced inhibition of the activity of succinic dehydrogenase, adenosine triphosphatase and acid phosphattase accompanied by cellular degeneration with complete absence of spermatocytes in the testis of rabbits. The biochemical and histological changes were more marked in the animals treated with the trivalent chromium than those exposed to hexavalent chromium and were progressive with the duration of exposure.     

Removal of hexavalent chromium in tannery wastewater by Bacillus cereus

Bacillus cereus was used to remove chromium (Cr(VI)) from medium containing tannery wastewater under different conditions. The maximum rate of Cr(VI) removal was attained at a temperature of 37?°C, pH of 7.0-9.0, and biomass of 20 g/L when the initial Cr(VI) concentration was less than 50?mg/L. Under the optimum conditions, the Cr(VI) in tannery wastewater was treated with each cellular component of B. cereus to detect its ability to reduce Cr(VI). The results showed that the removal rate of Cr(VI) for the cell-free extracts could reach 92.70%, which was close to that of the whole cells (96.85%), indicating that the Cr(VI) reductase generated by B.?cereus is primarily intracellular. Additionally, during continuous culture of the B. cereus, the strain showed good consecutive growth and removal ability. After treatment of 20?mg/L Cr(VI) for 48?h, the B. cereus was observed by SEM and TEM-EDX. SEM images showed that the B.?cereus used to treat Cr(VI) grew well and had a uniform cellular size. TEM-EDX analysis revealed large quantities of chromium in the B. cereus cells used to treat Cr(VI). Overall, the results presented herein demonstrate that B. cereus can be used as a new biomaterial to remove Cr(VI) from tannery wastewater.