Oral chromium(VI) does not affect the frequency of micronuclei in hematopoietic cells of adult mice and of transplacentally exposed fetuses

Chromium(VI) compounds are genotoxic in a variety of cellular systems. Their potential carcinogenicity is affected by toxicokinetic patterns restricting bioavailability to certain targets, and by metabolic pathways affecting interaction of chromate-derived reactive species with DNA. Epidemiological data indicate that chromium(VI) can be carcinogenic to the human respiratory tract following inhalation at doses that are only achieved in certain occupational settings. However, concern has been raised that adverse effects may also result from oral intake. In order to further explore this issue, we performed studies in BDF1 and Swiss mice of both genders and various age. Sodium dichromate dihydrate and potassium dichromate were administered either with the drinking water, up to a concentration of 500 mg chromium(VI)/l for up to 210 consecutive days, or in a single intragastric dose of 17.7 mg/kg body weight. Under these conditions, no increase of the micronucleus frequency was observed in either bone marrow or peripheral blood erythrocytes. Conversely, the same compounds induced a clastogenic damage following intraperitoneal injection, which by-passes detoxification mechanisms. In addition, due to the hypothesis that susceptibility may be increased during the period of embryogenesis, we treated pregnant mice, up to a concentration of 10mg chromium(VI)/l drinking water. There was no effect on the numbers of fetuses/dam and on body weight of fetuses. Again, no toxic or genotoxic effect was observed either in bone marrow of pregnant mice or in liver and peripheral blood of their fetuses. Thus, even at doses that largely exceed drinking water standards (up to 10,000 times) or by massive intragastric administration, chromium(VI) is not genotoxic to hematopoietic cells of either adult mice or transplacentally exposed fetuses. These conclusions are consistent with the poor toxicity and lack of carcinogenicity of oral chromium(VI), and are mechanistically explained by the high efficiency of chromium(VI) detoxification processes in the gastrointestinal tract.     

Oral chromium(VI) does not affect the frequency of micronuclei in hematopoietic cells of adult mice and of transplacentally exposed fetuses

Chromium(VI) compounds are genotoxic in a variety of cellular systems. Their potential carcinogenicity is affected by toxicokinetic patterns restricting bioavailability to certain targets, and by metabolic pathways affecting interaction of chromate-derived reactive species with DNA. Epidemiological data indicate that chromium(VI) can be carcinogenic to the human respiratory tract following inhalation at doses that are only achieved in certain occupational settings. However, concern has been raised that adverse effects may also result from oral intake. In order to further explore this issue, we performed studies in BDF1 and Swiss mice of both genders and various age. Sodium dichromate dihydrate and potassium dichromate were administered either with the drinking water, up to a concentration of 500 mg chromium(VI)/l for up to 210 consecutive days, or in a single intragastric dose of 17.7 mg/kg body weight. Under these conditions, no increase of the micronucleus frequency was observed in either bone marrow or peripheral blood erythrocytes. Conversely, the same compounds induced a clastogenic damage following intraperitoneal injection, which by-passes detoxification mechanisms. In addition, due to the hypothesis that susceptibility may be increased during the period of embryogenesis, we treated pregnant mice, up to a concentration of 10 mg chromium(VI)/l drinking water. There was no effect on the numbers of fetuses/dam and on body weight of fetuses. Again, no toxic or genotoxic effect was observed either in bone marrow of pregnant mice or in liver and peripheral blood of their fetuses. Thus, even at doses that largely exceed drinking water standards (up to 10,000 times) or by massive intragastric administration, chromium(VI) is not genotoxic to hematopoietic cells of either adult mice or transplacentally exposed fetuses. These conclusions are consistent with the poor toxicity and lack of carcinogenicity of oral chromium(VI), and are mechanistically explained by the high efficiency of chromium(VI) detoxification processes in the gastrointestinal tract.     

Hepatic and renal changes in albino rats caused by the administration of chromium (VI) in drinking water

The effects of sodium chromate administered in drinking water on liver and kidney of albino rats have been studied, through investigation of histological alterations and monitoring changes on serum urea levels and transaminases (GOT and GPT). Measurements have been done after 4, 8 and 12 weeks of treatment. The liquid intake of treated animals decreases with time. The amount of water drunk by treated rats is 1/2 of that drink by controls after 12 weeks. The histological alterations in liver and kidney are similar to those described elsewhere. Serum urea level is always higher in treated animals than in controls. GOT levels are similar in both treated and control rats, although always higher in the treated ones. GPT levels increase significantly after 12 weeks of treatment.     

Studies on the genotoxic effect of chromium oxide (Cr VI): Interaction with deoxyribonucleic acid in solution

Chromium is a toxic and carcinogenic compound widely distributed in environment. In the present study we have investigated the interaction of chromium oxide with DNA employing UV/vis and fluorescence spectroscopy as well as Circular dichroism, thermal denaturation, retardation polyacrylamide gel electrophoresis and DNA-cellulose affinity techniques. The results showed that the binding of chromium oxide to DNA is concentration dependent; at low concentration shows a little effect but ant higher concentrations (>100 μg/ml) reduced the absorbance at 260 and 210 nm producing hypochromicity. Also λ_max of the metal at 210, 260 and 350 nm was reduced. DNA chromophores quenched with the chromium oxide and decreased fluorescence emission intensity. Upon binding of the metal to DNA the elliplicity at positive extreme was decreased (275 nm) and increased the ellipticity of the DNA at negative extreme 245 nm. Thermal denaturation profile of DNA shifted to higher degrees upon chromium oxide binding which accompanied by hypochromicity. Also, affinity of chromium oxide to double stranded DNA was higher than single stranded DNA. From the result it is concluded that chromium oxide interacts with DNA via two modes of interaction inducing structural changes and DNA compaction evidence providing chromium oxide genotoxicity.     

In vivo nephrotoxicity induced in mice by chromium(VI)

The role of glutathione (GSH) and chromium (V) in chromium (VI)-induced nephrotoxicity in mice was investigated at 24 h after K2Cr(VI)2O7 ip injection. Nephrotoxicity was assessed by measurements of relative kidney weight and serum urea nitrogen. Cr(VI) nephrotoxicity was accompanied by decreased renal GSH and glutathione reductase (GSSG-R) levels. Pretreatment with buthionine sulfoximine, an inhibitor of GSH biosynthesis, enhanced Cr(VI)-induced nephrotoxicity, and remarkably diminished kidney GSH and GSSG-R levels. In contrast, pretreatment with glutathione methyl ester, a GSH-supplying agent, prevented Cr(VI) from exerting a harmful effect on mouse kidney and restored kidney GSH level. Administration of a Cr(V) compound, K3Cr(V)O8, induced much higher toxicity in mouse kidney than Cr(VI), but it failed to diminish renal GSH level. Another Cr(V) compound, Cr(V)-GSH complex, and Cr(III) nitrate did not cause a nephrotoxic effect in mice. The mechanism of Cr(VI)-induced nephrotoxicity was explained using GSH and Cr(V).     

An investigation into the genotoxic species generated during reduction of chromium(VI) by catechol(amine)s

Abstract

Chromium(VI) is a common occupational carcinogen.¹ The major carcinogenic and mutagenic species are proposed to be Cr(V) and Cr(IV) intermediates formed during the reduction of Cr(VI) to stable Cr(III) compounds,² although indirect evidence suggests that reactive oxygen species (ROS) may also be important.³ The reductions of Cr(VI) by some biological reductants (e.g. ascorbate) have been studied previously, and genotoxic Cr(IV/V) species have been detected.⁴ Another potential reductant in vivo is protein-bound DOPA, which is present on oxidised proteins at low steady-state concentrations prior to enzymatic breakdown.⁵ Recently, we have shown, by EPR spectroscopy, that the reactions of Cr(VI) with model DOPA compounds (catechol(amine)s), and with oxidised proteins themselves, generate several reactive intermediates, including Cr(V) complexes and organic radicals.⁶ Previous studies have proposed that ROS may also be produced during catechol(amine) oxidation.⁷ Here we describe studies of the interaction of DNA with the reactive species produced during the reductions of K₂Cr₂O₇ by catechol(amine)s.     

In vitro potential genotoxic effects of surface drinking water treated with chlorine and alternative disinfectants

A battery of in vitro short-term tests revealing different genetic end-points was set up in order to study surface-water genotoxicity after disinfection with different biocides: sodium hypochlorite (NaClO), chlorine dioxide (ClO(2)) and peracetic acid (PAA). The surface water both before and after disinfection was concentrated by adsorption on C(18) silica cartridges and the concentrates containing non-volatile organics were divided into different portions for chemical analyses and biological assays. The following in vitro tests were conducted on the water concentrates dissolved in DMSO: the Salmonella mutagenicity assay with S. typhimurium strains TA98 and TA100; the SOS Chromotest with Escherichia coli, the Microtox and Mutatox assays with Vibrio fischeri; and gene conversion, point mutation and mitochondrial DNA mutability assays with D7 diploid Saccharomices cerevisiae strain. The results show that the SOS Chromotest and the yeast assays are highly sensitive in detecting genotoxicity. The surface-water extracts were very often toxic to most of the test organisms considered, partially masking their potential mutagenic activity. Therefore, the assays with E. coli and with S. cerevisiae are more likely to show a mutagenic effect because these organisms are generally less sensitive to most toxic compounds. Among the tested disinfectants, NaClO and ClO(2) increased water genotoxicity, whereas PAA was able to slightly reduce raw water activity. However, because the organic compounds in the lake water varied with the season of the year, the disinfection processes, at times, both increased and decreased the raw water activity.     

In vitro interaction of mutagenic chromium (VI) with red blood cells

The interaction of mutagenic Cr(VI) with red blood cells has been studied by ESR spectroscopy. Signals of two Cr(V) species are observed almost immediately after contacting red cells with chromate(VI) aqueous solution at pH 7.4. The signal at go = 1.985, which decays within one hour, is attributed to a Cr(V) complex formed by glutathione due its reducing and chelating ability. The other signal at go = 1.979, which is distinctly more persistent, may indicate that some immobilization of the formed Cr(V) ions takes place on the macromolecular cell components, e.g. glycoproteins.     

In vivo singlet-oxygen generation in blood of chromium(VI)-treated mice

Although it is assumed from in vitro experiments that the generation of reactive oxygen species such as the singlet oxygen (1O2), the hydroxyl radical, and the superoxide anion are responsible for chromium(VI) toxicity/carcinogenicity, no electron spin resonance (ESR) evidence for the generation of 1O2 in vivo has been reported. In this study, we have employed an ESR spin-trapping technique with 2,2,6,6-tetramethyl-4-piperidone (TMPD), a specific 1O2 trap, to detect 1O2 in blood. The ESR spectrum of the spin adduct observed in the blood of mice given 4.8 mmol Cr(VI)/kg body weight exhibited the 1:1:1 intensity pattern of three lines with a hyperfine coupling constant A(N) = 16.08 G and a g-value = 2.0066. The concentration of spin adduct detected in the blood was 1.46 microM (0.1% of total Cr concentration). The adduct production was inhibited by the addition of specific 1O2 scavengers such as 1,4-diazabicyclo[2.2.2]octane and sodium azide to the blood. The results indicate that the spin adduct is nitroxide produced by the reaction of 1O2 with TMPD. This is the first report of ESR evidence for the in vivo generation of 1O2 in mammals by Cr(VI).     

Reduction of chromium(VI) in Chinese hamster V-79 cells

The cellular reduction of chromate(VI) was studied by electron spin resonance spectrometry. Incubation of Chinese hamster V-79 cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complex in a manner dependent on time (30 min-2 h) and concentration (50-500 microM). Following removal of extracellular chromate, the level of chromium(V) complex decreased quickly during the first hour but more slowly for the next hour, whereas the level of chromium(III) remained unchanged, indicating that chromium(III) is the ultimate ion of this metal in cells. Alkaline elution studies demonstrated that treatment of cells with Na2CrO4 induced DNA single-strand breaks that decreased quickly and DNA-protein crosslinks that persisted for 2 h after removal of this metal. These results suggest that the cellular levels of chromium(V) and chromium(III) may be associated with the formation of DNA damage induced by chromium (VI).     

Uptake of chromium(III) and chromium(VI) compounds in the yeast cell structure

The study presented in this article investigated the influence of different Cr(III) and Cr(VI) compounds in the cultivation medium on the uptake and localization of chromium in the cell structure of the yeast Candida intermedia. The morphology of the yeast cell surface was observed by the scanning electron microscopy. Results demonstrated that the growth inhibitory concentration of Cr(III) in the cultivation medium induced changes in the yeast cell shape and affected the budding pattern, while inhibitory concentration of Cr(VI) did not cause any visible effects on morphological properties of the yeast cells. The amount of total accumulated chromium in yeast cells and the distribution of chromium between the yeast cell walls and spheroplasts were determined by atomic absorption spectroscopy. No significant differences were found neither in total chromium accumulation nor in the distribution of chromium in yeast cell walls and spheroplasts between the two of Cr(VI) compounds. Conversely, substantial differences between Cr(III) compounds were demonstrated in the total uptake as well as the localization of chromium in yeast cells.     

Binding of chromium(VI) to histones: implications for chromium(VI)-induced genotoxicity

The first evidence has been obtained for Cr(VI) (chromate) binding to isolated calf thymus (CT) histones under physiological conditions (pH 7.4, Cl⁻ concentration 152 mM, 310 K). No significant Cr(VI) binding under the same conditions was observed for other extracellular and intracellular proteins, including albumin, apo-transferrin and G-actin, as well as for CT DNA. The mode of Cr(VI) binding to histones was studied by vibrational, electronic and X-ray absorption (X-ray absorption near-edge structure and X-ray absorption fine structure) spectroscopies and molecular mechanics calculations. A proposed binding mechanism includes electrostatic interactions of CrO₄ ²⁻ with protonated Lys and Arg residues of histones, as well as the formation of hydrogen bonds with the protein backbone. Similarly, Cr(VI) can bind to nuclear localization signals (typically, Lys- and Arg-rich fragments) of other nuclear proteins. Selective binding of Cr(VI) to newly synthesized nuclear proteins (including histones) in the cytoplasm is likely to be responsible for the active transport of Cr(VI) into the nuclei of living cells. Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Spectroscopic studies on the interaction of chromium (VI) and chromium (III) with keyhole limpet hemocyanin

The interactions of keyhole limpet hemocyanin (KLH) with chromium nitrate, potassium dichromate, and chromate were investigated using fluorescence, UV–vis absorption and circular dichroism (CD) spectroscopy under simulated physiological conditions. The experimental results showed that the different forms of chromium could quench the intrinsic fluorescence of KLH following a static quenching mechanism rather than by dynamic collision, which indicated that a Cr–KLH complex was formed. The Stern–Volmer quenching constants for the interaction indicated that the binding reaction of KLH with Cr(VI) was stronger the binding of KLH with Cr(III). The thermodynamic values for binding of Cr(VI) to KLH are ΔH > 0 and ΔS > 0. By contrast, the values for the interaction of Cr(III) with KLH are ΔH < 0 and ΔS < 0. The results of synchronous fluorescence, UV–vis absorption and CD spectroscopy showed that the α‐helical secondary structure and conformation of KLH were altered by different forms of chromium. Copyright © 2016 John Wiley & Sons, Ltd.     

Mechanism of chromium(VI) carcinogenesis

Since chromium(VI) is unreactive toward DNA under physiological conditions in vitro, the ability of carcinogenic chromium(VI) compounds to damage DNA depends on the presence of cellular redox components that reduce chromium(VI) to reactive species capable of interacting with DNA. We have examined the role of glutathione and hydrogen peroxide in chromium(VI)-induced DNA damage in vitro. Upon reaction with chromium(VI), glutathione produced chromium(V) and glutathione thiyl radical reactive intermediates, whereas hydrogen peroxide produced chromium(V) and hydroxyl radical. Reaction of DNA with chromium(VI) in the presence of glutathione resulted in binding of chromium and glutathione to DNA with little or no DNA strand breakage. Reaction of DNA with chromium(VI) in the presence of hydrogen peroxide produced the 8-hydroxydeoxy-guanosine adduct and extensive DNA strand breakage in the absence of significant Cr-DNA adduct formation. These results suggest that the nature of chromium(VI)-induced DNA damage will be strongly dependent on reactive intermediates such as chromium(V), glutathione thiyl radical, and hydroxyl radical, produced by cellular components active in chromium(VI) metabolism. In order to assess the ability of chromium(VI)-induced DNA damage to affect the normal template function of DNA, we investigated the effects of chromium(VI) on steady-state mRNA levels of various genes in chick embryo liver in vivo, and compared the effects to the levels of DNA damage observed. Chromium(VI) induced DNA-protein and DNA interstrand cross-links in chick embryo liver in vivo and suppressed the induction of 5-aminolevulinic acid synthase and cytochrome P-450 mRNA expression by porphyrinogenic drugs. In contrast, chromium(VI) increased the basal levels of expression of these two inducible genes, but had little or no effect on the expression of the constitutive albumin, β-actin, and conalbumin genes. Comparison of the time course of formation and repair of DNA damage with that of changes in gene expression suggests that chromium(VI) may form a mono-adduct prior to formation of DNA cross-links, and that chromium(VI)-induced DNA lesions may target certain classes of genes and lead to changes in their expression.     

On the interaction of compounds of chromium(VI) with hydrogen peroxide. A study of chromium(VI) and (V) peroxides in the acid-basic pH range

A computational study of chromium(VI) and (V) peroxides, which exhibit important genotoxic and mutagenic activity, is reported. Energies and equilibrium geometries for [Cr^VI(O)(O₂)₂(OH)]⁻, [Cr^VI(O)(O₂)₂(OH₂)], [Cr^VI(O)(O₂)₂(py)], [Cr^VI(OH)(O₂)₂(OH₂)]⁺, [Cr^V(O)(O₂)₂(OH₂)]⁻ and species were calculated using molecular mechanics calculations (MMFF94 and MM⁺), quantum calculations with semi-empirical methods (RHF and UHF/PM3) and density functional theory (pBP86/DN* or pBP/DN* and B3LYP/6-31G(d). Equilibrium geometries for the compounds [Cr^V(O₂)₃(OH)]²⁻ and [Cr^V(O₂)₄]³⁻ were determined by molecular mechanics. Vibrational frequencies, standard thermodynamic quantities and electronic spectra were calculated using B3LYP/6-31G(d). The structural relationship between all these species and an explanation of the formation of peroxo species in the acid-basic pH range are given. An experimental study of peroxo species in basic medium was also performed (synthesis, X-ray powder diffraction patterns and infrared spectra of the peroxo complexes isolated) but did not confirm the existence of a tri-peroxo complex in the solid phase.     

Lack of cytogenetic effects in mice or mutations in Salmonella receiving sodium fluoride

We have examined the possible effect of fluoride intake on chromosome damage. There was no evidence of increased frequency of chromosomal aberration in bone marrow or testis cells of mice with either 50 ppm fluoride intake over several generations or 100 ppm intake for 6 weeks compared to animals drinking distilled water. Fluoride was not found to be mutagenic in a widely used bacterial mutagenesis assay over a range of 0.1 to as high as 2000 μg fluoride per plate.     

Cytotoxic and genotoxic effects of methotrexate in germ cells of male Swiss mice

Methotrexate (MTX) is an anti-metabolite drug widely used in the treatment of neoplastic disorders, rheumatoid arthritis and psoriasis. Developed as an analogue of folic acid, it inhibits purine and pyrimidine synthesis that accounts for its therapeutic efficacy as well as for its toxicities. MTX has narrow therapeutic index and its toxicity has been reported in various organ systems including gastrointestinal, haematologic and central nervous system. The objective of the present study is to investigate the germ cell toxicity induced by MTX in male Swiss mice. MTX was administered intraperitoneally (ip) at the doses of 5, 10, 20 and 40mg/kg to mice (20-25g) weekly once (wk) for 5 and 10 weeks. The animals were sacrificed 1 week after receiving the last treatment of MTX. The germ cell toxicity was evaluated using testes weight (wt), sperm count, sperm head morphology, sperm comet assay, histology, TUNEL and halo assay in testis. MTX treatment significantly reduced the sperm count and increased the occurrence of sperm head abnormalities in a dose dependent manner. It induced the testicular toxicity as evident from the histology of testis. Sperm comet, TUNEL and halo assay in testis also revealed significant DNA damage after MTX treatment. On the basis of the present study, it can be concluded that MTX induced germ cell toxicity in mice.     

Lack of cytogenetic effects in mice or mutations in Salmonella receiving sodium fluoride.

We have examined the possible effect of fluoride intake on chromosome damage. There was no evidence of increased frequency of chromosomal aberration in bone marrow or testis cells of mice with either 50 ppm fluoride intake over several generations or 100 ppm intake for 6 weeks compared to animals drinking distilled water. Fluoride was not found to be mutagenic in a widely used bacterial mutagenesis assay over a range of 0.1 to as high as 2000 microgram fluoride per plate.