Carcinogenicity of hexavalent chromium.

Hexavalent chromium (Cr(VI)), a commonly used industrial metal, is a well known human lung carcinogen. Epidemiology and animal studies suggest that the particulate Cr(VI) compounds, specifically the water insoluble compounds, are the more potent carcinogens; however, the carcinogenic mechanism remains unknown. Here we summarize recent Cr(VI)-induced human tumour, in vivo, cell culture and in vitro studies and put the data into context with three major paradigms of carcinogenesis: multistage carcinogenesis, genomic instability, and epigenetic modifications. Based on these studies, we propose a mechanism for chromate carcinogenesis that is primarily driven by the genomic instability paradigm.

Benzo[a]pyrene cytotoxicity in right whale (Eubalaena glacialis) skin, testis and lung cell lines.

The North Atlantic right whale (NARW) is one of the most endangered great whales. The NARW population consists of only about 300 individuals and is reproducing at an insufficient rate. There is growing concern about the potential effects of environmental contaminants on the reproductive and overall health of NARW. High contaminant burdens can accumulate in tissues of great whales but toxicological studies of their effects are limited due to legal, logistical and ethical restrictions and specific in vitro models are critically needed. Cell lines from NARW skin and internal organs were previously created in our laboratory. In this study, skin, testis and lung primary fibroblast cell lines were exposed to benzo[a]pyrene (BP) as part of a multi-chemical toxicity testing project in NARW. Cells were exposed for 24-72 h to 10 nM-10 microM BP dissolved in dimethylsulfoxide. Cytotoxicity was measured with a clonogenic assay using standard methods. Some cytotoxicity was observed after 24 h, the highest concentration (10 microM BP) resulting in 77, 74 and 51 percent relative survival in testis, skin and lung cells, respectively, and indicating a higher cytotoxicity in the lung (p < 0.05). After 48 and 72-h exposure, 10 microM BP resulted in 24 and 3, 74 and 27, and 42 and 23 percent relative survival in testis, skin and lung cells, respectively. Cytotoxicity significantly increased with exposure time in all three tissues (p < 0.05 for skin and p < 0.01 for lung and testis), suggesting metabolic activation of BP in the three organs. Fibroblast cytotoxicity observed in the testis was higher than that observed either in the skin or lung after 48 h (p < 0.01) and was close to 100% after 72 h, warranting further investigation of the potential effects of PAHs on reproductive health.

Glutathione S-transferase M1 and T1 genetic polymorphisms, alcohol consumption and breast cancer risk.

Alcohol consumption has been inconsistently associated with breast cancer risk. Recent studies suggest that genetic polymorphisms of glutathione S-transferases (GSTs) may modify this relation. To determine if breast cancer risk is associated with GSTM1 and GSTT1 genetic polymorphisms, and to evaluate the effect modification between GST genotypes and alcohol consumption in the risk of breast cancer, we conducted a case-control study in the state of Connecticut in the period 1998 and 2001. Cases were histologically confirmed, incident breast cancer patients in New Haven County, CT. Controls were randomly selected from women histologically confirmed to be without breast cancer. The study results show that, while GSTM1 genotypes were not associated with breast cancer risk, GSTT1-null genotype was associated with a significant 90% increased risk for postmenopausal women (OR=1.9, 95% CI 1.2-3.0). Analysis by GST genotypes and alcohol consumption shows that GSTM1A ever-drinking women had a 2.5-fold (OR=2.5, 95% CI 1.1-5.5) increased risk of breast cancer compared to the GSTM1A never-drinkers, and the risk increases with duration and daily amount of alcohol consumption. Postmenopausal women with GSTT1-null genotype, who consumed a lifetime of >250 kg of spirit-equivalents, had an almost seven-fold increased risk (OR=6.8, 95% CI 1.4-33.9), and drinking commencing at younger ages appears to carry a higher risk. An OR of 8.2 (95% CI 1.2-57.4) was observed for those with GSTM1A, and GSTT1-null genotypes who had consumed a lifetime of >250 kg of spirit-equivalents. In conclusion, alcohol consumption may increase breast cancer risk among those who carry susceptible GST genotypes.

Does aluminum exposure of pregnant animals lead to accumulation in mothers or their offspring?

There is concern that environmental and dietary aluminum (Al) might cause developmental toxicity. To better understand this concern, we reviewed published studies which administered Al compounds to pregnant animals and measured accumulation of Al in mother, fetus, or born offspring. A total of 7 studies were identified which administered Al during gestation and evaluated fetal accumulation. Another 7 studies administered Al at least until birth and then evaluated accumulation in mothers and/or pups. These 14 studies included 4 different Al compounds (hydroxide, chloride, lactate, and citrate) administered by 4 different routes (gavage, feed, intraperitoneal injection, and subcutaneous injection) with total doses ranging from 13.5 to 8,400 mg/kg. Fetal Al levels were not increased in 6 of 7 studies and pup Al levels were not increased in 4 of 5 studies in which they were measured. Maternal Al levels were increased in some studies, but there was no consistent pattern of organ-specific accumulation and several positive studies were contradicted by subsequent reports from the same laboratory. Placental levels were increased in 6 of 9 studies and were greater than corresponding fetal levels. The weight of evidence in these studies suggests that environmental and dietary Al exposures are unlikely to pose risks of Al accumulation to pregnant animals or their fetuses.

Induction of apoptotic cell death by particulate lead chromate: differential effects of vitamins C and E on genotoxicity and survival.

Certain hexavalent chromium compounds are documented human carcinogens. Exposure of cells to particulate forms of chromium results in cell-enhanced dissolution of particles in the extracellular microenvironment and chronic production of chromium oxyanions, which are taken up by the cell through an anion transport system and are genotoxic and clastogenic. It was previously shown that apoptosis is the mode of cell death of nearly all of the Chinese hamster ovary cells (CHO-AA8 cell line), which die after high-dose, short-term treatments with soluble sodium chromate. In this report the mode of cell killing by particulate lead chromate and of low-dose continuous treaments of soluble sodium chromate designed to mimic conditions of ionic chromate uptake after lead chromate exposure was examined. CHO-AA8 cells were treated for 24 hr with doses of sodium chromate or lead chromate which cause a 50% decrease in survival in colony-forming effeciency assays. Longer treatments (up to 72 hr) at the same doses did not decrease survival further than the 24-hr exposure. Morphological changes indicative of apoptosis, as well as internucleosomal DNA fragmentation, were detectable by 24 hr after treatment with lead chromate or soluble sodium chromate. All of the cells killed by treatments with lead chromate particles underwent apoptosis as the mode of cell death and this was accurately modeled in cell culture by continuous treatments with low-dose soluble sodium chromate. Exposure of cells to hexavalent chromium compounds causes a spectrum of DNA damage which can be selectively altered by pretreatment of cells with antioxidant vitamins prior to chromium exposure. Here we show that ascorbate and alpha-tocopherol markedly inhibited the chromosomal aberrations induced by both particulate and soluble chromate compounds, even though chromium adduct levels were not decreased by either vitamin pretreatment. Cell survival assays showed that ascorbate, but not alpha-tocopherol, protected cells from apoptosis induced by sodium chromate. The results differentiate chromium-induced apoptosis from both chromosomal damage and adduct levels and suggest that other lesions sensitive to ascorbate but not tocopherol are the proximal inducing signal for chromium-induced apoptosis.

Chromium(III) picolinate produces chromosome damage in Chinese hamster ovary cells.

Chromium(III) complexes currently being sold as dietary supplements were tested for their ability to cause chromosomal aberrations in Chinese hamster ovary cells. Complexes were tested in soluble and particulate forms. Chromium picolinate was found to produce chromosome damage 3-fold to 18-fold above control levels for soluble doses of 0.050, 0.10, 0.50, and 1.0 mM after 24 h treatment. Particulate chromium picolinate doses of 8.0 micrograms/cm2 (corresponding to a 0.10 mM solublized dose) and 40 micrograms/cm2 (0.50 mM) produced aberrations 4-fold and 16-fold above control levels, respectively. Toxicity was measured as a decrease in plating efficiency relative to controls. The above treatments produced > or = 86% survival for all doses except 1.0 mM chromium picolinate, which produced 69 +/- 10% survival. Chromium nicotinate, nicotinic acid, and chromium(III) chloride hexahydrate did not produce chromosome damage at equivalent nontoxic doses. Damage was inferred to be caused by the picolinate ligand because picolinic acid in the absence of chromium was clastogenic. Data are evaluated in terms of their relevance to human exposure based on pharmacokinetic modeling of tissue accumulation and are discussed in terms of literature reporting toxic effects of picolinic acid.

Cell-enhanced dissolution of carcinogenic lead chromate particles: the role of individual dissolution products in clastogenesis.

Lead chromate induces chromosomal damage as a result of extracellular dissolution producing solubilized chromium and lead and we show here that the dissolution process is greatly accelerated by the presence of cells. We have sought to determine which of these ions is involved in lead chromate-induced clastogenicity. Cell-mediated extracellular dissolution of particulate lead chromate resulted in the accumulation of both solubilized chromium and solubilized lead, reaching concentrations in the extracellular medium of 15 and 1.9 microM respectively and reaching concentrations inside the cell of 2700 and 97 microM respectively. Both the extracellular and intracellular accumulation of chromium was time dependent and both the solubilized lead and chromium increased proportionately from a lower dose to a higher dose. Exposing cells to water soluble sodium chromate under conditions which produced similar time-dependent intracellular concentrations of chromium also produced a similar amount and spectrum of chromosome damage as lead chromate. In contrast, exposure to lead glutamate resulted in intracellular lead levels 438-times higher than those produced by lead chromate, but produced no chromosome damage. A higher dose of lead glutamate was weakly clastogenic, but it induced a different spectrum of chromosomal aberrations than lead chromate. Pretreatment of cells with vitamin E had no effect on the uptake of chromium, but reduced both sodium chromate- and lead chromate-induced clastogenesis by 54-93%. Vitamin E pretreatment did not affect lead glutamate-induced clastogenesis. The results of this study indicate that although lead(II) is weakly clastogenic at high doses, hexavalent chromium is the proximate clastogen in lead chromate-induced clastogenesis. Additionally, this is the first report that pretreatment of cells with vitamin E can block clastogenesis induced by particulate chromates.

Induction of internucleosomal DNA fragmentation by carcinogenic chromate: relationship to DNA damage, genotoxicity, and inhibition of macromolecular synthesis.

Hexavalent chromium (Cr) compounds are respiratory carcinogens in humans and animals. Treatment of Chinese hamster ovary cells with 150 and 300 microM sodium chromate (Na2CrO4) for 2 hr decreased colony-forming efficiency by 46 and 92%, respectively. These treatments induced dose-dependent internucleosomal fragmentation of cellular DNA beyond 24 hr after chromate treatment. This fragmentation pattern is characteristic of apoptosis as a mechanism of cell death. These treatments also induced an immediate inhibition of macromolecular synthesis and delayed progression of cells through S-phase of the cell cycle. Cell growth (as evidenced by DNA synthesis) was inhibited for at least 4 days and transcription remained suppressed for at least 32 hr. Many of the cells that did progress to metaphase exhibited chromosome damage. Chromate caused the dose-dependent formation of DNA single-strand breaks and DNA-protein cross-links, but these were repaired 8 and 24 hr after removal of the treatment, respectively. In contrast, Cr-DNA adducts (up to 1/100 base-pairs) were extremely resistant to repair and were still detectable even 5 days after treatment. Compared with other regions of the genome, DNA-protein cross-links and Cr adducts were preferentially associated with the nuclear matrix DNA of treated cells, which was 4.5-fold enriched in actively transcribed genes. Chromium adducts, formed on DNA in vitro at a similar level to that detected in nuclear matrix DNA, arrested the progression of a DNA polymerase in a sequence-specific manner, possibly through the formation of DNA-DNA cross-links.(ABSTRACT TRUNCATED AT 250 WORDS)

An inhibitor of nuclear scaffold protease blocks chemical transformation of fibroblasts.

A nuclear scaffold (NS) protease has previously been implicated in production of the M(r) 46,000 ATP-binding protein in NS (which may acquire nucleoside triphosphatase activity and participate in nucleocytoplasmic transport) by cleavage of a subset of lamins A/C. In a preceding paper (G. Clawson, L. Norbeck, C. Hatem, C. Rhodes, P. Amiri, J. McKerrow, S. Patierno, and G. Fiskum, Cell Growth & Differ., 3: 827-838), this NS protease was identified as a novel, Ca(2+)-regulated serine protease, which was found only in the NS and which appears to represent a unique multicatalytic protease complex. Based upon its predominantly chymotrypsin-like substrate preference, a peptide-chloromethylketone inhibitor (succinyl-AAPF-chloromethylketone, AAPFcmk) was identified. AAPFcmk showed a KI = 56 nM for the NS protease versus 1.4 microM for the endoplasmic reticulum activity. Treatment of C3H/10T1/2 mouse embryo fibroblast cells with 1 microM AAPFcmk produced effects which were confined to the nuclear (and to a lesser extent the endoplasmic reticulum) compartment. In this report, we examine the effects of the AAPFcmk inhibitor on cellular transformation and growth. Growth of C3H/10T1/2 cells was decreased by 34% and 56% at 25 microM and 50 microM AAPFcmk, respectively. Growth inhibition occurred without any major change in DNA content distribution, suggesting effects throughout the cell cycle. Growth inhibition was not observed at lower (< or = 10 microM) concentrations, which decreased transformation of C3H/10T1/2 fibroblasts in a dose-dependent manner by up to 90%, even at femtomolar concentrations of AAPFcmk (in the absence of growth inhibition). Inclusion of irrelevant inhibitors was without affect.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of lead chromate clastogenesis by ascorbate: relationship to particle dissolution and uptake.

Chromium metal salts are considered to be human carcinogens, especially the salts of low solubility. Lead chromate, a highly insoluble chromium metal salt, has been shown to be tumorigenic, genotoxic and clastogenic. In this study, the roles of particle-cell contact, particle dissolution and particle uptake in the clastogenic activity of lead chromate were investigated. Using Pb51CrO4 it was found that lead chromate particles (1.2 microns mean diameter, -28 mV surface charge) were slightly soluble in water; solubility increased 2-fold when particles were incubated in culture medium, but was not increased further by the addition of serum. The extracellular concentration of chromium increased 7-fold when lead chromate was incubated in the presence of Chinese hamster ovary (CHO) cells compared with culture medium alone. The intracellular concentration of ionic chromium increased in a dose-dependent manner following exposure of CHO cells to clastogenic doses of lead chromate reaching estimated levels as high as 1.2 mM per cell. Treatment of cells with lead chromate particles in the presence of a nontoxic dose of vitamin C blocked uptake of ionic chromium and eliminated the clastogenic activity of the particles. Transmission electron microscopy showed that lead chromate particles were internalized by CHO cells in phagocytic vacuoles in as little as 1 h; internalization was unaffected by co-treatment with vitamin C. It was demonstrated that particle-cell contact was required for lead chromate-induced clastogenesis. These data show that although phagocytic particle uptake occurs, particle-cell contact and extracellular dissolution are responsible for the clastogenic activity of lead chromate. These data also demonstrate that the genotoxicity of particulate hexavalent chromates can be blocked by vitamin C.

DNA damage induced by carcinogenic lead chromate particles in cultured mammalian cells.

Particulate lead chromate is a highly water-insoluble cytotoxic and carcinogenic agent, but its mechanism of action remains obscure. We investigated its effects on DNA damage in CHO cells after a 24-h exposure using alkaline or neutral filter elution and cytogenetic studies. Concentrations (0.08, 0.4 and 0.8 micrograms/cm2), which reduced the colony-forming efficiency of CHO cells to 94, 50 and 10%, respectively, produced dose-dependent DNA single-strand breaks and DNA-protein crosslinks, but no DNA double-strand breaks or DNA-DNA crosslinks were observed. The single-strand breaks were absent from cells given a 24-h recovery period after removal of the treatment medium, even though most of the particles remained adhered to cells and to the culture dish. In contrast, both the DNA-protein crosslinks and chromosomal aberrations persisted even after the 24-h recovery period. These results suggest that the mechanism of the particle-induced early DNA single-strand breaks may be different from DNA-protein crosslinks and the lesions leading to chromosomal aberrations, or alternatively, that the repair of single-strand breaks is more efficient than the repair of DNA-protein crosslinks in the unavoidable continuing presence of carcinogen. These results also suggest that the chromosome damage may be related to the persistent DNA-protein crosslinks, and further confirm the genotoxic activity of carcinogenic lead chromate particles.

Clastogenicity of lead chromate particles in hamster and human cells.

Several insoluble compounds of chromium, such as lead chromate, are respiratory carcinogens in experimental animals and suspected to be so in humans. Lead chromate induces neoplastic transformation in cultured cells but the mechanism of genotoxicity is unknown. We examined the effect of lead chromate on the integrity of chromosomes of Chinese hamster ovary (CHO) and human foreskin fibroblasts (HFF) after a 24-h exposure. At 0.4 microgram/cm2, 0.8 microgram/cm2, 2 microgram/cm2 and 8 microgram/cm2 lead chromate particles reduced survival of CHO cells to 86%, 62%, 2% and less than 1% respectively. These concentrations induced a dose-dependent 4-19-fold increase in the percent metaphases with damage. The HFF cells exhibited higher sensitivity in both cytotoxicity and clastogenicity. The spectrum of damage observed for both cell types was primarily achromatic lesions affecting one or both chromatids. To test for particle dissolution effects, CHO cells were treated for 24 h with either clarified medium that had been incubated for 24 h with lead chromate particles, or clarified medium that had been pre-conditioned by CHO cells treated with lead chromate particles for 24 h. No damage was detected in these cells, indicating that extracellular dissolution into ionic lead and chromate did not contribute to the genotoxicity. This is consistent with a previous study in which scanning electron micrographs illustrated internalization of the particles. These results suggest that clastogenesis may be a mechanism for lead chromate induced carcinogenesis.