Jane Lee

Comparative toxicity of arsenic metabolites in human bladder cancer EJ-1 cells.

The human bladder is one of the primary target organs for arsenic-induced carcinogenicity, and arsenic metabolites in urine have been suspected to be directly involved in carcinogenesis. Thioarsenicals are commonly found in human and animal urine and are also considered to be highly toxic arsenic metabolites. The present study was performed to gain insight into the toxicity and accumulation of arsenic species found in urine, including arsenate (iAs(V)), arsenite (iAs(III)), monomethylarsonic acid (MMA(V)), monomethylmonothioarsonic acid (MMMTA(V)), dimethylarsinic acid (DMA(V)), dimethylarsinous acid (DMA(III)), dimethylmonothioarsinic acid, (DMMTA(V)), and dimethyldithioarsinic acid (DMDTA(V)) in human bladder cancer EJ-1 cells. The order of cytotoxicity of these arsenic compounds in EJ-1 human bladder cancer cells was DMA(III), DMMTA(V) > iAs(III) ≫ iAs(V) > MMMTA(V) > MMA(V), DMA(V), and DMDTA(V), indicating that the sulfur-containing DMMTA(V) was among the most toxic arsenic compounds similar to trivalent DMA(III). We further characterized the DNA damage, generation of highly reactive oxygen species (hROS), and expression of proteins p21 and p53 in cells after exposure to iAs(III), DMA(III), and DMMTA(V). Cellular exposure to DMMTA(V) resulted in reduced protein expression of p53 and p21, increased DNA damage, and increased intracellular hROS (hydroxyl radical). In contrast, iAs(III) significantly increased the protein expression of p21 and p53 and did not increase the hROS at the IC(50). Intracellular glutathione (GSH) was reduced by 60% after exposure to DMA(III) or DMMTA(V), suggesting that DMMTA(V) causes cell death through oxidative stress. In contrast, GSH levels increased in cells exposed to iAs(III), and hROS only increased after a long exposure to iAs(III). Our findings demonstrate that DMMTA(V) may be one of the most toxicologically potent arsenic species, relevant to arsenic-induced carcinogenicity in the urinary bladder.

Evidence for toxicity differences between inorganic arsenite and thioarsenicals in human bladder cancer cells.

Arsenic toxicity is dependent on its chemical species. In humans, the bladder is one of the primary target organs for arsenic-induced carcinogenicity. However, little is known about the mechanisms underlying arsenic-induced carcinogenicity, and what arsenic species are responsible for this carcinogenicity. The present study aimed at comparing the toxic effect of DMMTA(V) with that of inorganic arsenite (iAs(III)) on cell viability, uptake efficiency and production of reactive oxygen species (ROS) toward human bladder cancer EJ-1 cells. The results were compared with those of a previous study using human epidermoid carcinoma A431 cells. Although iAs(III) was known to be toxic to most cells, here we show that iAs(III) (LC(50)=112 microM) was much less cytotoxic than DMMTA(V) (LC(50)=16.7 microM) in human bladder EJ-1 cells. Interestingly, pentavalent sulfur-containing DMMTA(V) generated a high level of intracellular ROS in EJ-1 cells. However, this was not observed in the cells exposed to trivalent inorganic iAs(III) at their respective LC(50) dose. Furthermore, the presence of N-acetyl-cysteine completely inhibited the cytotoxicity of DMMTA(V) but not iAs(III), suggesting that production of ROS was the main cause of cell death from exposure to DMMTA(V), but not iAs(III). Because the cellular uptake of iAs(III) is mediated by aquaporin proteins, and because the resistance of cells to arsenite can be influenced by lower arsenic uptake due to lower expression of aquaporin proteins (AQP 3, 7 and 9), the expression of several members of the aquaporin family was also examined. In human bladder EJ-1 cells, mRNA/proteins of AQP3, 7 and 9 were not detected by reverse transcription polymerase chain reaction (RT-PCR)/western blotting. In A431 cells, only mRNA and protein of AQP3 were detected. The large difference in toxicity between the two cell lines could be related to their differences in uptake of arsenic species.

Attenuation of DNA damage-induced p53 expression by arsenic: a possible mechanism for arsenic co-carcinogenesis.

Inhibition of DNA repair processes has been suggested as one predominant mechanism in arsenic co‐genotoxicity. However, the underlying mode of action responsible for DNA repair inhibition by arsenic remains elusive. To further elucidate the mechanism of repair inhibition by arsenic, we examined the effect of trivalent inorganic and methylated arsenic metabolites on the repair of benzo(a)pyrene diol epoxide (BPDE)–DNA adducts in normal human primary fibroblasts and their effect on repair‐related protein expression. We observed that monomethylarsonous acid (MMAIII) was the most potent inhibitor of the DNA repair. MMAIII did not change the expression levels of some key repair proteins involved upstream of the dual incision in the global nucleotide excision repair (NER) pathway, including p48, XPC, xeroderma pigmentosum complementation group A (XPA), and p62‐TFIIH. However, it led to a marked impairment of p53 induction in response to BPDE treatment. The abrogated p53 expression translated into reduced p53 DNA‐binding activity, suggesting a possibility of downregulating downstream repair genes by p53. A p53‐null cell line failed to exhibit the inhibitory effect of MMAIII on NER, implicating a role for p53 in the NER inhibition by MMAIII. Further investigation revealed that MMAIII dramatically inhibited p53 phosphorylation at serine 15, implying that MMAIII destabilized p53 by inhibiting its phosphorylation. Because p53 is required for proficient global NER, our data suggest that arsenic inhibits NER through suppressing p53 induction in response to DNA damage in cells with normal p53 gene expression.

Genetic predisposition to the cytotoxicity of arsenic: the role of DNA damage and ATM

Arsenic is a pervasive cytotoxin and carcinogen in the environment. Although its mode of action has yet to be fully elucidated, oxidative DNA damage has been suggested. A series of DNA repair‐defective human and hamster cell lines associated with sensitivity to oxidative agents were examined for their response to arsenic‐induced cytotoxicity. Only the Ataxia telangiectasia (AT) cells displayed a marked hypersensitive response (greater than twofold). The protective role of the ATM protein was confirmed by the normal response to arsenic displayed by AT cells expressing wild‐type ATM. Although the ATM protein plays a pivotal role in response to DNA double‐strand breakage, none of the other cell lines with defects in double‐strand break repair displayed a similar hypersensitivity. Further examination indicated that concentrations of sodium arsenite as high as 1 mg/l do not generate significant levels of double‐strand breaks. Our data suggest that the ATM protein functions in an important but different capacity in the cellular response to arsenic toxicity than it does in response to agents that generate double‐strand breaks, such as ionizing radiation. Furthermore, the lack of hypersensitivity to arsenic displayed by the other cell lines calls into question the hypothesis that DNA damage is a significant factor in arsenic cytotoxicity.

Detection of DNA adducts of benzo[a]pyrene using immunoelectrophoresis with laser-induced fluorescence analysis of A549 cells

Detection of benzo[a]pyrene diol epoxide (BPDE)-damaged DNA in a human lung carcinoma cell line (A549) has been performed using free zone affinity capillary electrophoresis with laser-induced fluorescence (LIF). Using BPDE as a model carcinogenic compound, the speed, sensitivity and specificity of this technique was demonstrated. Under free zone conditions, an antibody bound adduct was baseline-resolved from an unbound adduct in less than 2 min. The efficiencies of separation were in excess of 6 x 10(5) and 1 x 10(6) plates per meter for the antibody-bound and unbound adducts, respectively. Separation using a low ionic strength buffer permitted the use of a high electric field (830 V/cm) without the loss of resolving power. Using LIF detection, a concentration detection limit of roughly 3 x 10(-10) M was achieved for a 90-mer oligonuleotide containing a single BDPE. The use of formamide in the incubation buffer to enhance denaturing of DNA did not affect the stability of the complex between the antibody and the adducts. Using a fluorescently labeled BPDE-modified DNA adduct probe, a competitive assay was established to determine the levels of BPDE-DNA adducts in A549 cells.

Arsenite and its mono- and dimethylated trivalent metabolites enhance the formation of benzo[a]pyrene diol epoxide-DNA adducts in Xeroderma pigmentosum complementation group A cells.

Recently, inorganic arsenite (iAs(III)) and its mono- and dimethylated metabolites have been examined for their interference with the formation and repair of benzo[a]pyrene diol epoxide (BPDE)-induced DNA adducts in human cells (Schwerdtle, ., Walter, I., and Hartwig, A. (2003) DNA Repair 2, 1449 - 1463). iAs(III) and monomethylarsonous acid (MMA(III)) were found to be able to enhance the formation of BPDE-DNA adducts, whereas dimethylarsinous acid (DMA(III)) had no enhancing effect at all. The anomaly manifested by DMA(III) prompted us to further investigate the effects of the three trivalent arsenic species on the formation of BPDE-DNA adducts. Use of a nucleotide excision repair (NER)-deficient Xeroderma pigmentosum complementation group A cell line (GM04312C) allowed us to dissect DNA damage induction from DNA repair and to examine the effects of arsenic on the formation of BPDE-DNA adducts only. At concentrations comparable to those used in the study by Schwerdtle et al., we found that each of the three trivalent arsenic species was able to enhance the formation of BPDE-DNA adducts with the potency in a descending order of MMA(III) > DMA(III) > iAs(III), which correlates well with their cytotoxicities. Similar to iAs(III), DMA(III) modulation of reduced glutathione (GSH) or total glutathione S-transferase (GST) activity could not account for its enhancing effect on DNA adduct formation. Additionally, the enhancing effects elicited by the trivalent arsenic species were demonstrated to be highly time-dependent. Thus, although our study made use of short-term assays with relatively high doses, our data may have meaningful implications for carcinogenesis induced by chronic exposure to arsenic at low doses encountered environmentally.

Elevation of Cellular BPDE Uptake by Human Cells: A Possible Factor Contributing to Co-Carcinogenicity by Arsenite

Background Arsenite (iAsIII) can promote mutagenicity and carcinogenicity of other carcinogens. Considerable attention has focused on interference with DNA repair by inorganic arsenic, especially the nucleotide excision repair (NER) pathway, whereas less is known about the effect of arsenic on the induction of DNA damage by other agents. Objectives We examined how arsenic modulates DNA damage by other chemicals. Methods We used an NER-deficient cell line to dissect DNA damage induction from DNA repair and to examine the effects of iAsIII on the formation of benzo[a]pyrene diol epoxide (BPDE)–DNA adducts. Results We found that pretreatment with iAsIII at subtoxic concentrations (10 μM) led to enhanced formation of BPDE–DNA adducts. Reduced glutathione levels, glutathione S-transferase activity and chromatin accessibility were also measured after iAsIII treatment, but none of these factors appeared to account for the enhanced formation of DNA adducts. However, we found that pretreatment with iAsIII increased the cellular uptake of BPDE in a dose-dependent manner. Conclusions Our results suggest that iAsIII enhanced the formation of BPDE–DNA adducts by increasing the cellular uptake of BPDE. Therefore, the ability of arsenic to increase the bioavailability of other carcinogens may contribute to arsenic co-carcinogenicity.

Factors influencing the removal of thymine glycol from DNA in γ-irradiated human cells

Abstract The toxic and mutagenic effects of ionizing radiation are believed to be caused by damage to cellular DNA. We have made use of a novel immunoassay for thymine glycol to examine the removal of this lesion from the DNA of irradiated human cells. Because of the sensitivity of the assay, we have been able to keep the radiation doses at or below the standard clinical dose of 2 Gy. Our initial observations indicated that although removal of thymine glycol is >80% complete by 4 h post-irradiation with 2 Gy, there is a lag of 30-60 min before