In this study, we used the IST method, which measures DNA radicals directly, to investigate the ability of inorganic arsenite to induce ODD in arsenite methylation–deficient and –competent cells. A major advantage of the IST method is that it converts unstable DNA radicals induced by reactive oxygen species into stable nitrone adducts allowing for direct measurement of DNA radicals and avoiding sample preparation–induced artifactual ODD, making results more reliable than previous methods (27
). Furthermore, we used biologically relevant levels of arsenite exposure: chronic exposure to inorganic arsenic levels in the 5.0 μM range in drinking water has been associated with oxidative stress in humans (43
In our initial experiments, we found ODD to occur in TRL1215 cells, which can methylate arsenic, but not in RWPE-1 cells, which cannot, even though both cell lines were exposed to arsenite concentrations and durations sufficient to cause an acquired malignant phenotype in vitro (in the present work) and instill the ability when inoculated to cause formation of malignant tumors in immunodeficient mice (3
). An inhibitor of arsenic methylation, sodium selenite (40
), abolished arsenite-induced ODD and the acquisition of an in vitro cancer phenotype in arsenic methylation–competent TRL1215 cells. These observations were fortified by our observation that arsenic methylation–competent (As3mt-
transduced) UROtsa/F35 cells showed much more ODD upon arsenic exposure than its parental UROtsa cell line and acquired a transformed phenotype in vitro in much less time. Hence, it appears that arsenic methylation is obligatory for ODD in some cells and hastens the acquisition of cancer phenotype. However, because cells unable to methylate arsenic can still acquire an arsenic-induced cancer phenotype in vitro over time, it is likely that arsenic is also carcinogenic by mechanisms that neither require biomethylation nor ODD. It is possible that multiple mechanisms may account for carcinogenesis by arsenic even within the same cell line and that the speed with which a cancer phenotype is acquired depends on the number of mechanisms at play.
Chronic exposure to low levels of sodium arsenite caused a remarkable, but delayed, increase in ODD only in arsenic methylation–competent cells. Accumulating evidence indicates that the methylation of arsenic is not a detoxifying event and that arsenic biomethylation may produce highly toxic compounds with genotoxic potential (13
). Although inorganic arsenic can stimulate reactive oxygen species production, trivalent methylated arsenic compounds appear to stimulate the production of reactive oxygen species more efficiently than inorganic arsenic compounds (13
). Indeed, ex vivo evidence indicates that dimethylarsinous acid is an indirect genotoxin that forms hydroxyl radicals through an unknown mechanism (44
). In addition to directly increasing reactive oxygen species, arsenic compounds might also induce ODD indirectly by inhibiting important detoxifying enzymes. Generally speaking, trivalent methylated arsenicals are more potent inhibitors of enzymes when compared with inorganic forms, possibly because of higher affinities for critical thiol groups (45
). It is important to note that high concentrations of inorganic arsenic that are lethal to the cells can acutely induce ODD in cells without arsenic biomethylation, like RWPE-1 cells (data not shown), but the relevance of finding ODD in dead or dying cells to cancer is undoubtedly limited.
Several striking, mutually supportive similarities were evident in the temporal pattern of ODD generation in the arsenic methylation–competent cells (TRL1215 and UROtsa/F35) in this study. First, both the human and the rat cells showed a delay of several weeks before the onset of ODD [ (7
)]. Second, both cell lines showed a rapid drop-off in levels of ODD at about the point in time they acquired a cancer phenotype [ (7
)]. The reasons for both phenomena are unclear. The delay may involve buildup of a key toxic metabolite, such as a methylated arsenic compound. Initially, we suspected that the precipitous loss of ODD might be attributed to adaptation to arsenic; adaptive increases in arsenic transport–related gene expression (eg, GSTP1
) and in glutathione levels are not uncommon, all of which promote arsenic efflux from exposed cells (40
). Chronic arsenic exposure also typically increases the expression of stress-response genes such as NFE2L2
, and metallothionein (39
). We had suspected that a differential adaptive response might account for the loss of ODD in TRL1215 cells, but we found these genes to also be overexpressed in RWPE-1 cells (which show no ODD) at about the time of arsenic-induced malignant transformation, so this gene expression pattern is not likely to be an adaptation caused by ODD loss. In any event, by the time of the precipitous drop in ODD, it appears that sufficient damage had already occurred to hasten malignant transformation in methylation-competent cells.
Some limitations of this study have already been discussed. It is unknown why arsenic biomethylation–competent cells show a delayed onset of ODD with arsenic exposure or why there is a precipitous drop near the point of acquired cancer phenotype. Furthermore, the observed ODD, although consistently linked to more rapid acquisition of cancer phenotype, is not linked to precise precipitating events (ie, mutations) that would drive cells more rapidly toward malignancy. This is an unproven assumption. Only four cell lines were used in this work, two of which are isogenic with the exception of the transduction of As3mt. Methylated arsenicals were never actually measured, and it is only assumed that they were differentially produced based on prior work. Cell lines exposed here to arsenite in vitro were not directly used in in vivo xenograft studies but were assumed to be transformed based on in vitro phenotype and timing relative to earlier experiments. In vivo testing in arsenic methylase knockout mice would be critical to confirm our hypothesis. In vitro cell models of cancer may not be strictly concordant in timing, and so on, with much more complicated formation of tumors in vivo.
In summary, chronic exposure to inorganic arsenic consistently caused a delayed increase in ODD, but only in cells able to methylate arsenic, which was then precipitously lost at about the same time that these methylation-competent cells acquired a cancer phenotype in vitro. Arsenite-induced ODD appeared to accelerate acquisition of cancer phenotype in vitro and only required the transduction of a single gene, As3mt
. Human data are emerging that clearly show that polymorphisms in AS3MT
exist that affect arsenical methylation patterns (50
) and thereby may alter susceptibility to carcinogenesis. AS3MT
-null humans are not known, but As3mt
knockout mice have very recently been introduced (51
). Although inorganic arsenicals have not yet been tested for carcinogenic effects in these genetically altered mice, this clearly should be a high priority. AS3MT
polymorphism analysis may one day provide a metric of human susceptibility to arsenical carcinogenesis at critical target sites.