Kirk T. Kitchin

Oxidative stress as a possible mode of action for arsenic carcinogenesis.

Many modes of action for arsenic carcinogenesis have been proposed, but few theories have a substantial mass of supporting data. Three stronger theories of arsenic carcinogenesis are production of chromosomal abnormalities, promotion of carcinogenesis and oxidative stress. This article presents the oxidative stress theory along with some supporting experimental data. In the area of which arsenic species is causually active, recent data have suggested that trivalent methylated arsenic metabolites, particularly monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), have a great deal of biological activity. Some evidence now indicates that these trivalent, methylated, and relatively less ionizable arsenic metabolites may be unusually capable of interacting with cellular targets such as proteins and even DNA. Thus for inorganic arsenic, oxidative methylation followed by reduction to trivalency may be a activation, rather than a detoxification pathway. This would be particularly true for arsenate. In forming toxic and carcinogenic arsenic species, reduction from the pentavalent state to the trivalent state may be as or more important than methylation of arsenic.

The role of protein binding of trivalent arsenicals in arsenic carcinogenesis and toxicity.

Three of the most plausible biological theories of arsenic carcinogenesis are protein binding, oxidative stress and altered DNA methylation. This review presents the role of trivalent arsenicals binding to proteins in arsenic carcinogenesis. Using vacuum filtration based receptor dissociation binding techniques, the lifetimes of unidentate (<1s), bidentate (1-2min) and tridentate (1-2h) arsenite containing peptide binding complexes were estimated. According to our experimental data some of the protein targets to which arsenite may bind in vivo include tubulin, poly(ADP-ribose)polymerase (PARP-1), thioredoxin reductase, estrogen receptor-alpha, arsenic(+3)methyltransferase and Keap-1. Arsenite binding to tubulin can lead to several of the genetic effects observed after arsenic exposures (aneuploidy, polyploidy and mitotic arrests). Among many other possible arsenite binding sites are rat hemoglobin, the DNA repair enzyme xeroderma pigmentosum protein A (XPA), and other C2H2, C3H and C4 zinc finger proteins including members of the steroid receptor superfamily (e.g. glucocorticoid receptor). Macromolecules to which arsenite does not bind to include calf thymus DNA, mixed Type II-A histones and bovine H3/H4 histone. Although all six tested arsenicals released iron from ferritin, radioactive arsenite did not bind to the protein horse ferritin.

Arsenic-Induced Carcinogenesis—Oxidative Stress as a Possible Mode of Action and Future Research Needs for More Biologically Based Risk Assessment

Exposure to inorganic arsenic (iAs) induces cancer in human lungs, urinary bladder, skin, kidney, and liver, with the majority of deaths from lung and bladder cancer. To date, cancer risk assessments for iAs have not relied on mechanistic data, as we have lacked sufficient understanding of arsenics pharmacokinetics and mode(s) of carcinogenic action (MOA). Furthermore, while there are vast amounts of toxicological data on iAs, relatively little of it has been collected using experimental designs that efficiently support development of biologically based dose-response (BBDR) models and subsequently risk assessment. This review outlines an efficient approach to the development of a BBDR model for iAs that would reduce uncertainties in its cancer risk assessment. This BBDR-based approach is illustrated by using oxidative stress as the carcinogenic MOA for iAs but would be generically applicable to other MOAs. Six major research needs that will facilitate BBDR model development for arsenic-induced cancer are (1) MOA research, which is needed to reduce the uncertainty in risk assessment; (2) development and integration of the pharmacodynamic component (MOA) of the BBDR model; (3) dose-response and extrapolation model selection; (4) the determination of internal human speciated arsenical concentrations to improve physiologically based pharmacokinetic (PBPK) models; (5) animal models of arsenic carcinogenesis; and (6) the determination of the low dose human relationship for death from cancer, particularly in lungs and urinary bladder. The major parts of the BBDR model are arsenic exposure, a physiologically based pharmacokinetic model, reactive species, antioxidant defenses, oxidative stress, cytotoxicity, growth factors, transcription factors, DNA damage, chromosome damage, cell proliferation, mutation accumulation, and cancer. The BBDR model will need to be developed concurrently with data collection so that model uncertainties can be identified and addressed through an iterative process of targeted additional research.

Dimethylarsinic acid treatment alters six different rat biochemical parameters: relevance to arsenic carcinogenesis.

In a previous study, we found that sodium arsenite increased hepatic ornithine decarboxylase (ODC) activity and hepatic heme oxygenase (HO) activity, but did not cause any DNA damage in adult female rat liver or lung, suggesting that arsenite may be a promoter of carcinogenesis. In this study sodium arsenate, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA) were administered orally in equitoxic doses to adult female rats at 21 and 4 h prior to sacrifice. DNA damage (DD), cytochrome P450 content (P450), glutathione content (GSH), ODC, serum alanine aminotransferase (ALT) and HO were measured in liver and/or lung tissue. At 60 mg/kg in rat liver, sodium arsenate increased hepatic HO fivefold. MMA decreased ALT at 226 mg/kg, decreased ALT and GSH at 679 mg/kg and also increased P450 at 679 mg/kg in rat liver. DMA decreased ALT and hepatic GSH and increased hepatic HO at 387 mg/kg. In the lung, DMA decreased ODC at both 129 and 387 mg/kg. DD in lung tissue was significantly higher at 387 mg/kg DMA, demonstrating organ specific DNA damage. The biochemical effects and the inferred oncologic potential of the four major forms of arsenic (arsenate, arsenite, MMA and DMA) differ dramatically. The inorganic forms (arsenate and arsenite) are similar to each other (both good HO inducers); the methylated organic forms of arsenic (MMA and DMA) also share a similar pattern of biochemical effects (decreased GSH and ALT, increased P450). All six of the biochemical parameters studied were altered by DMA in either rat liver or lung.

Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin.

Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. With ascorbic acid the rate of iron release from HLF by DMA(V) was intermediate (3.37 nM/min, P<0.05) and by DMA(III) was much higher (16.3 nM/min, P<0.001). No pBR322 plasmid DNA damage was observed from in vitro exposure to arsenate (iAs(V)), arsenite (iAs(III)), monomethylarsonic acid (MMA(V)), monomethylarsonous acid (MMA(III)) or DMA(V) alone. DNA damage was observed following DMA(III) exposure; coexposure to DMA(III) and HLF caused more DNA damage; considerably higher amounts of DNA damage was caused by coexposure of DMA(III), HLF and ascorbic acid. Diethylenetriaminepentaacetic acid (an iron chelator), significantly inhibited DNA damage. Addition of catalase (which can increase Fe(2+) concentrations) further increased the plasmid DNA damage. Iron-dependent DNA damage could be a mechanism of action of human arsenic carcinogenesis.

Teratogenicity of cyclophosphamide in a coupled microsomal activating/embryo culture system

Abstract Using the coupled microsomal activating/embryo culture system, in vitro experiments were performed to establish the role of metabolism in the embryo toxicity and teratogenicity of cyclophosphamide. Cyclophosphamide in the coupled microsomal activating/embryo culture system produced characteristic morphological lesions as well as a general inhibition of embryo and yolk sac growth. Increasing concentrations of NADPH in the presence of microsomes and cyclophosphamide produced progressively greater responses. These effects did not occur when microsomes and NADPH were present in the serum medium for the first 2 hours of incubation followed by one washing and then culturing of the conceptuses from hr 2 to hr 48 in a medium containing cyclophosphamide alone. Cytochrome P-450-depleted microsomes did not bioactivate cyclophosphamide to teratogenic or toxic metabolites. The results indicate that cytochrome P-450-dependent microsomal metabolism of cyclophosphamide is required for the embryotoxic and teratogenic effects observed in vitro.

Arsenite, but not cadmium, induces ornithine decarboxylase and heme oxygenase activity in rat liver: relevance to arsenic carcinogenesis.

Sodium arsenite and cadmium chloride, were administered orally to adult female rats at 21 and 4 h prior to sacrifice. Liver, lung, skin and urinary bladder were the tissues studied. DNA damage, cytochrome P450, glutathione content (GSH), ornithine decarboxylase (ODC), serum alanine aminotransferase and heme oxygenase activity were measured. Sodium arsenite increased rat hepatic ODC activity at 1.6 and 24.6 mg/kg and hepatic heme oxygenase activity at 8.2 and 24.6 mg/kg, but did not cause any DNA damage. Cadmium chloride did not affect any of the six parameters tested. These findings suggest that sodium arsenite may be a promoter rather than an initiator of carcinogenesis.

Further development of rodent whole embryo culture: solvent toxicity and water insoluble compound delivery system.

In order to study the in vitro embryotoxicity and dysmorphogenesis of water insoluble compounds, solvents or chemical delivery systems of low toxicity and teratogenicity to the developing embryo must be found. Therefore, day 10.5 rat embryos were cultured for 2 days in whole rat serum containing 0.1, 0.5 and 2.5 vol.% of ethyl alcohol, dimethylsulfoxide, acetone, Tween 80, corn oil and 10% acetone/90% corn oil. No adverse effects occurred with any of the solvents at the 0.1% concentration level. At 0.5% ethyl alcohol and Tween 80 significantly reduced embryonic growth and increased the incidence of embryonic abnormalities. With the exception of corn oil and acetone/corn oil, embryos cultured in media containing 2.5% of various solvents failed to grow, did not differentiate, and died during the culture period. Corn oil suspended in rat serum by use of ultrasound was non-toxic even at concentrations of 2.5% and 10%. Growth parameters of embryos cultured in serum containing corn oil were indistinguishable from controls and overall morphogenesis was good (particularly at 2.5%). The order of increasing embryotoxicity and dysmorphogenesis of the studied liquids was corn oil less than acetone/corn oil less than dimethylsulfoxide less than ethyl alcohol, acetone less than Tween 80. Any of the 4 water miscible solvents (at 0.1%) or a sonicated suspension of corn oil in serum (up to 2.5%) met the criteria of a non-toxic and non-teratogenic water insoluble compound delivery system for in vitro embryo culture.

An integrated pharmacokinetic and pharmacodynamic study of arsenite action. 1. Heme oxygenase induction in rats

Rat heme oxygenase (HO) activity was used as a specific (among forms of arsenic) and sensitive biomarker of effect for orally administered sodium arsenite in rats. Time course studies showed that HO was induced in rat liver from 2 to 48 h in both rat liver and kidney. Hepatic and renal inorganic arsenic (iAs) concentrations were high at times preceding a high degree of HO induction. At times following pronounced HO induction, tissue dimethylarsinic acid concentrations were high. Dose-response studies of arsenite showed substantial HO induction in liver at doses of 30 micromol/kg and higher and in the kidney at doses of 100 micromol/kg and higher. Doses of 10 (in liver) and of 30 micromol/kg (in kidney) sodium arsenite given by gavage did not significantly induce rat HO activity. Speciation of tissue total arsenic into iAs, methylarsonic acid (MMA), and dimethylarsinic acid (DMA) permits us to link tissue iAs and HO enzyme induction. There was a linear relationship between tissue inorganic arsenic (iAs) concentration and tissue HO in individual rats (r(2) = 0.780 in liver and r(2) = 0.797 in kidney). Nonlinear relationships were observed between administered arsenite dose and either liver or kidney iAs concentration. Overall, there was a sublinear relationship between administered arsenite and biological effect in rats. Teratogenesis Carcinog. Mutagen. 19:385-402, 1999. Published 1999 Wiley-Liss, Inc.

Myeloperoxidase-induced genomic DNA-centered radicals

Myeloperoxidase (MPO) released by activated neutrophils can initiate and promote carcinogenesis. MPO produces hypochlorous acid (HOCl) that oxidizes the genomic DNA in inflammatory cells as well as in surrounding epithelial cells. DNA-centered radicals are early intermediates formed during DNA oxidation. Once formed, DNA-centered radicals decay by mechanisms that are not completely understood, producing a number of oxidation products that are studied as markers of DNA oxidation. In this study we employed the 5,5-dimethyl-1-pyrroline N-oxide-based immuno-spin trapping technique to investigate the MPO-triggered formation of DNA-centered radicals in inflammatory and epithelial cells and to test whether resveratrol blocks HOCl-induced DNA-centered radical formation in these cells. We found that HOCl added exogenously or generated intracellularly by MPO that has been taken up by the cell or by MPO newly synthesized produces DNA-centered radicals inside cells. We also found that resveratrol passed across cell membranes and scavenged HOCl before it reacted with the genomic DNA, thus blocking DNA-centered radical formation. Taken together our results indicate that the formation of DNA-centered radicals by intracellular MPO may be a useful point of therapeutic intervention in inflammation-induced carcinogenesis.