Elizabeth T. Snow

Arsenic and heavy metal contamination of vegetables grown in Samta village, Bangladesh

Drinking of arsenic (As) contaminated well water has become a serious threat to the health of many millions in Bangladesh. However, the implications of contamination of agricultural soils from long-term irrigation with As-contaminated groundwater for phyto-accumulation in food crops, and thence dietary exposure to As, and other metals, has not been assessed previously in Bangladesh. Various vegetables were sampled in Samta village in the Jessore district of Bangladesh, and screened for As, Cd, Pb, Cu and Zn by inductively coupled plasma emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). These local food products are the basis of human nutrition in this region and of great relevance to human health. The results revealed that the individual vegetables containing the highest mean As concentrations microg x g(-1)) are snake gourd (0.489), ghotkol (0.446), taro (0.440), green papaya (0.389), elephant foot (0.338) and Bottle ground leaf (0.306), respectively. The As concentration in fleshy vegetable material is low. In general, the data show the potential for some vegetables to accumulate heavy metals with concentrations of Pb greater than Cd. Some vegetables such as bottle ground leaf, ghotkol, taro, eddoe and elephant foot had much higher concentrations of Pb. Other leafy and root vegetables, contained higher concentrations of Zn and Cu. Bioconcentration factors (BCF) values, based on dry weight, were below 1 for all metals. In most cases, BCF values decreased with increasing metal concentrations in the soil. From the heavily As-contaminated village in Samta, BCF values for As in ladies finger, potato, ash gourd, brinjal, green papaya, ghotkol and snake gourd were 0.001, 0.006, 0.006, 0.014, 0.030, 0.034 and 0.038, respectively. Considering the average daily intake of fresh vegetables per person per day is only 130 g, all the vegetables grown at Samta had Pb concentrations that would be a health hazard for human consumption. Although the total As in the vegetables was less than the recommended maximum intake of As, it still provides a significant additional source of As in the diet.

Metal carcinogenesis: Mechanistic implications

Cancer epidemiology has identified several metal compounds as human carcinogens. Recent evidence suggests that carcinogenic metals induce genotoxicity in a multiplicity of ways, either alone or by enhancing the effects of other agents. This review summarizes current information on the genotoxicity of arsenic, chromium, nickel, beryllium and cadmium compounds and their possible roles in carcinogenesis. Each of these metals is distinct in its primary modes of action; yet there are several mechanisms induced by more than one metal, including: the induction of cellular immunity and oxidative stress, the inhibition of DNA metabolism and repair and the formation of DNA- and/or protein-crosslinks.

Toxicity and Carcinogenicity of Nickel Compounds

The toxicity and carcinogenicity of nickel compounds are considered in three broad categories: (1) systemic toxicology, (2) molecular toxicology, and (3) carcinogenicity. The systemic toxicity of nickel compounds is examined based upon human and animal studies. The major organs affected are discussed in three categories: (1) kidney, (2) immune system, and (3) other organs. The second area of concentration is molecular toxicology, which will include a discussion of the chemistry of nickel, its binding to small and large molecular weight ligands, and, finally, its cellular effects. The third major area involves a discussion of the carcinogenicity and genotoxicity of nickel compounds. This section focuses on mechanisms, using studies conducted in vivo and in vitro. It also includes a discussion of the assessment of the carcinogenicity of nickel compounds.

A comparison of trace element concentrations in cultured and wild carp (Cyprinus carpio) of Lake Kasumigaura, Japan

The concentrations of 13 elements were determined in the muscle, liver, intestine, kidney, and gonads of cultured and wild carp caught at two sites in Lake Kasumigaura, Japan, between September 1994 and September 1995. Despite having a reputation for being heavily polluted, the carp were not heavily burdened with metals. Our results suggest that despite their dietary differences, the wild and cultured fish were accumulating and distributing metals in the same manner and that aquaculture practices are not increasing metal concentrations in these fish. Metal concentrations were lowest in muscle, and did not exceed established quality standards for fish. The differences in metal concentrations between cultivated and wild carp are negligible and should pose no health problems for consumers of either type of fish.

Arsenic toxicity is enzyme specific and its affects on ligation are not caused by the direct inhibition of DNA repair enzymes

The molecular mechanism of arsenic toxicity is believed to be due to the ability of arsenite [As(III)] to bind protein thiols. Numerous studies have shown that arsenic is cytotoxic at micromolar concentrations. Micromolar As can also induce chromosomal damage and inhibit DNA repair. The mechanism of arsenic-induced genotoxicity is very important because arsenic is a human carcinogen, but not a mutagen, and there is a need to establish recommendations for safe levels of As in the environment. We have measured the dose-response for arsenic inhibition of several purified human DNA repair enzymes, including DNA polymerase beta, DNA ligase I and DNA ligase III and have found that most enzymes, even those with critical SH groups, are very insensitive to As. Many repair enzymes are activated by millimolar concentrations of As(III) and/or As(V). Only pyruvate dehydrogenase, one of eight purified enzymes examined so far, is inhibited by micromolar arsenic. In contrast to the purified enzymes, treatment of human cells in culture with micromolar arsenic produces a significant dose-dependent decrease in DNA ligase activity in nuclear extracts from the treated cells. However, the ligase activity in extracts from untreated cells is no more sensitive to arsenic than the purified enzymes. Our results show that direct enzyme inhibition is not a common toxic effect of As and that only a few sensitive enzymes are responsible for arsenic-induced cellular toxicity. Thus, arsenic-induced co-mutagenesis and inhibition of DNA repair is probably not the result of direct enzyme inhibition, but may be an indirect effect caused by As-induced changes in cellular redox levels or alterations in signal transduction pathways and consequent changes in gene expression.

Effect of arsenic on transcription factor AP-1 and NF-kB DNA binding activity and related gene expression

Both acute (24 h) and chronic (10-20 week) exposure of human fibroblast cells to low dose sodium arsenite (As(III)) significantly affects activating protein-1 (AP-1) and nuclear factor kappa B (NF-kappa B) DNA binding activity. Short-term treatment with 0.1-5 microM As(III) up-regulates expression of c-Fos and c-Jun and the redox regulators, thioredoxin (Trx) and Redox factor-1 (Ref-1) and activates both AP-1 and NF-kappa B binding. Chronic exposure to 0.1 or 0.5 microM As(III) decreased c-Jun, c-Fos and Ref-1 protein levels and AP-1 and NF-kappa B binding activity, but increased Trx expression. Short term exposure to phorbol 12-myristate 13-acetate (TPA), a phorbol ester tumour promoter, or hydrogen peroxide (H(2)O(2)) also activates AP-1 and NF-kappa B binding. However, pre-treatment with As(III) prevents this increase. These results suggest that As(III) may alter AP-1 and NF-kappa B activity, in part, by up-regulating Trx and Ref-1. The different effects of short- versus long-term As(III) treatment on acute-phase response to oxidative stress reflect changes in the expression of Ref-1, c-Fos and c-Jun, but not Trx.

An Escherichia coli plasmid-based, mutational system in which supF mutants are selectable: Insertion elements dominate the spontaneous spectra

A new system is described to determine the mutational spectra of mutagens and carcinogens in Escherichia coli; data on a limited number (142) of spontaneous mutants is presented. The mutational assay employs a method to select (rather than screen) for mutations in a supF target gene carried on a plasmid. The E. coli host cells (ES87) are lacI- (am26), and carry the lacZ delta M15 marker for alpha-complementation in beta-galactosidase. When these cells also carry a plasmid, such as pUB3, which contains a wild-type copy of supF and lacZ-alpha, the lactose operon is repressed (off). Furthermore, supF suppression of lacIam26 results in a lactose repressor that has an uninducible, lacIS genotype, which makes the cells unable to grow on lactose minimal plates. In contrast, spontaneous or mutagen-induced supF- mutations in pUB3 prevent suppression of lacIam26 and result in constitutive expression of the lactose operon, which permits growth on lactose minimal plates. The spontaneous mutation frequency in the supF gene is approximately 0.7 and approximately 1.0 x 10(-6) without and with SOS induction, respectively. Spontaneous mutations are dominated by large insertions (67% in SOS-uninduced and 56% in SOS-induced cells), and their frequency of appearance is largely unaffected by SOS induction. These are identified by DNA sequencing to be Insertion Elements; IS1 dominates, but IS4, IS5, gamma-delta and IS10 are also obtained. Large deletions also contribute significantly (19% and 15% for -SOS and +SOS, respectively), where a specific deletion between a 10 base pair direct repeat dominates; the frequency of appearance of these mutations also appears to be unaffected by SOS induction. In contrast, SOS induction increases base pairing mutations (13% and 27% for -SOS and +SOS, respectively). The ES87/pUB3 system has many advantages for determining mutational spectra, including the fact that mutant isolation is fast and simple, and the determination of mutational changes is rapid because of the small size of supF.

Effects of chromium(III) on DNA replication in vitro

A number of metal compounds are important environmental carcinogens; however, the molecular mechanisms of metal-induced genotoxicity are not yet understood. Chromium, for example, is substantially mutagenic in vivo and has been shown to decrease the DNA replication fidelity in vitro. But the mechanism of chromium-induced mutagenesis is unkown and the role of replication fidelity in chromium-induced carciogenesis is unclear. We have used in vitro DNA replication assays to investigate the effects of chromium ions on DNA polymerase activity preliminary to studying their role in chromium-induced mutagenesis. Biologically active M13mp2 DNA was replicated with purified DNA polymerases in the presence of micromolar amounts of chromium with or without the normal divalent cation, magnesium. Nucleotide incorporation kinetics were determined and sequence specific pausing was analyzed by primer-extension. Our results have demonstrated an unexpected polymerase activation by low (0.5–5.0 μm) concentrations of chromium(III) although higher concentrations of chromium are increasingly inhibitory. The increased incorporation seem at low chromium(III) concentrations is the result of increased enzyme processivity and is not polymerase specific. The possible relationship between processivity and metal-ion mutagenesis is discussed.

Replication across O6-Methylguanine by Human DNA Polymerase β in Vitro INSIGHTS INTO THE FUTILE CYTOTOXIC REPAIR AND MUTAGENESIS OF O6-METHYLGUANINE

Replication in vivo across unrepaired O6-methylguanine (m6dG) lesions by mammalian DNA polymerase β (pol β) during short patch repair may contribute to the cytotoxicity and mutagenesis of m6dG. We have employed in vitro steady state kinetic analysis to investigate the replication of oligonucleotide templates containing site-specific m6dG by human pol β. Our results show that m6dG is a strong but not absolute block to replication by pol β. pol β exhibits mixed kinetic discrimination during overall replication across dG and m6dG. pol β preferentially inserts dTMP rather than dCMP opposite m6dG. However, pol β extends from the dC-m6dG base pair more efficiently than from the dT-m6dG base pair. This is in strong contrast to other polymerases such as the exonuclease-deficient Klenow fragment of Escherichia coli DNA polymerase I (exo−KF) that preferentially extends dT-m6dG by a factor of 10 over dC-m6dG. When both insertion and extension are considered, pol β has a 15-fold overall preference for incorporation of the mutagenic substrate dTTP rather than the nonmutagenic substrate dCTP during replication across m6dG. This suggests that pol β, in concert with the T:G-specific thymine DNA glycosylase, may be intricately involved in the futile cytotoxic repair induced by m6dG. Our results also suggest that replication across m6dG by pol β may contribute to m6dG-induced G → A transition mutations.

Metal ions and carcinogenesis

Metals are essential for the normal functioning of living organisms. Their uses in biological systems are varied, but are frequently associated with sites of critical protein function, such as zinc finger motifs and electron or oxygen carriers. These functions only require essential metals in minute amounts, hence they are termed trace metals. Other metals are, however, less beneficial, owing to their ability to promote a wide variety of deleterious health effects, including cancer. Metals such as arsenic, for example, can produce a variety of diseases ranging from keratosis of the palms and feet to cancers in multiple target organs. The nature and type of metal-induced pathologies appear to be dependent on the concentration, speciation, and length of exposure. Unfortunately, human contact with metals is an inescapable consequence of human life, with exposures occurring from both occupational and environmental sources. A uniform mechanism of action for all harmful metals is unlikely, if not implausible, given the diverse chemical properties of each metal. In this chapter we will review the mechanisms of carcinogenesis of arsenic, cadmium, chromium, and nickel, the four known carcinogenic metals that are best understood. The key areas of speciation, bioavailability, and mechanisms of action are discussed with particular reference to the role of metals in alteration of gene expression and maintenance of genomic integrity.