Howard A. Zaren

Induction of glutathione S-transferase π as a bioassay for the evaluation of potency of inhibitors of benzo(a)pyrene-induced cancer in a murine model

There is a growing need for short‐term and cost‐effective bioassay to assess the efficacy of potential chemo‐preventive agents. We report that the induction of glutathione (GSH) S‐transferase π (mGSTP1‐1) by a chemo‐preventive agent can be used as a reliable marker to assess its efficacy in retarding chemical carcinogenesis induced by benzo(a)pyrene (BP), which is a widespread environmental pollutant and believed to be a risk factor in human chemical carcinogenesis. This conclusion is based on 1) the relative contribution of mGSTP1‐1 of the liver and forestomach of female A/J mice in the detoxification of the ultimate carcinogenic metabolite of BP, (+)‐anti‐7,8‐dihydroxy‐9,10‐oxy‐7,8,9,10‐ tetrahydrobenzo(a)pyrene [(+)‐anti‐BPDE]; and 2) a positive correlation between the induction of hepatic and forestomach mGSTP1‐1 by 5 naturally occurring organosulfides (OSCs) from garlic (diallyl sulfide, diallyl disulfide, diallyl trisulfide, dipropyl sulfide and dipropyl disulfide) and their effectiveness in preventing BP‐induced forestomach neoplasia in mice. In the liver, the combined contribution of other GSTs in the detoxification of (+)‐anti‐BPDE was far less than the contribution of mGSTP1‐1 alone. Likewise, in the forestomach, the contribution of mGSTP1‐1 far exceeded the combined contribution of other GSTs. Studies on the effects of OSCs against BP‐induced forestomach neoplasia revealed a good correlation between their chemo‐preventive efficacy and their ability to induce mGSTP1‐1 expression in the liver (r = −0.89; p < 0.05) as well as in the forestomach (r = −0.97; p < 0.05). Our results suggest that the induction of mGSTP1‐1 may be a reliable marker for evaluating the efficacy of potential inhibitors of BP‐induced cancer in a murine model. Int. J. Cancer 73:897–902, 1997.

Mechanism of differential efficacy of garlic organosulfides in preventing benzo(a)pyrene-induced cancer in mice

The mechanism of differential efficacies of diallyl sulfide (DAS), diallyl disulfide (DADS), diallyl trisulfide (DATS), dipropyl sulfide (DPS) and dipropyl disulfide (DPDS) in preventing benzo(a)pyrene (BP)-induced cancer in mice has been investigated by determining their effects on the enzymes of BP activation/inactivation pathways. With the exception of DATS, treatment of mice with other organosulfides (OSCs) caused a small but significant increase (37-44%) in hepatic ethoxyresorufin O-deethylase (EROD) activity. However, the forestomach EROD activity did not differ significantly between control and treated groups. Only DAS treatment caused a modest but statistically significant reduction (about 25%) in pulmonary EROD activity. These results suggest that while reduction of EROD activity may, at least in part, contribute to the DAS-mediated inhibition of BP-induced lung cancer, anticarcinogenic effects of OSCs against BP-induced forestomach carcinogenesis seems to be independent of this mechanism. Treatment of mice with DAS, DADS and DATS resulted in a significant increase, as compared with control, in both hepatic (3.0-, 3.2- and 4.4-fold, respectively) and forestomach (1.5-, 2.7- and 2.7-fold, respectively) glutathione transferase (GST) activity toward anti-7beta,8alpha-dihydroxy-9alpha,10alpha-oxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BPDE), which is the ultimate carcinogen of BP. The pulmonary GST activity was not increased by any of the OSCs. Even though epoxide hydrolase (EH) activity was differentially altered by these OSCs, a correlation between chemopreventive efficacy of OSCs and their effects on EH activity was not apparent. The results of the present study suggest that differences in the ability of OSCs to modulate GST activity toward anti-BPDE may, at least in part, account for their differential chemopreventive efficacy against BP-induced cancer in mice.

Gender-related differences in susceptibility of A/J mouse to benzo[a]pyrene-induced pulmonary and forestomach tumorigenesis

Benzo[a]pyrene (BP) is a suspected human carcinogen and is known to produce tumors in the lung and forestomach of mice. Glutathione (GSH) S-transferases (GST) play a major role in the detoxification of the ultimate carcinogen of BP, (+)-anti-7,8-dihydroxy-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene ((+)-anti-BPDE). Previous studies have shown gender-related differences in the expression of GST isoenzymes in mice. The present study was designed to test the hypothesis whether gender-related differences in the expression of GST isoenzymes can affect the susceptibility of mice to BP-induced lung and forestomach tumorigenesis. The expression of pi class isoenzyme mGSTP1-1, which is highly efficient in the detoxification of (+)-anti-BPDE, was approximately 3.0- and 1.5-fold higher in the liver and forestomach of male A/J mouse, respectively, as compared with the female. The levels of other major GST isoenzymes, mGSTA3-3 (alpha class), mGSTM1-1 (mu class) and mGSTA4-4 (alpha class), were also significantly higher in the liver of the male mouse as compared with the female. While pulmonary mGSTP1-1 expression did not differ significantly between male and female A/J mice, the expression of mGSTA3-3, mGSTM1-1 and mGSTA4-4 was significantly higher (1.4-4.0-fold) in the lung of the male A/J mouse as compared with the female. At lower concentrations of BP (0.5 mg BP/mouse), the tumor incidence/multiplicity was significantly higher in the lung as well as in the forestomach of female mice as compared with male mice. For example, while 30% of the female mice developed pulmonary tumors 26 weeks after the first 0.5 mg BP administration, none of the male mice had tumors in their lungs. At higher doses of BP (1.5 mg BP/mouse), however, this differential was either abolished or relatively less pronounced. Our results suggest that up to a certain threshold of BP exposure the levels of GST isoenzymes may be an important determinant of susceptibility to BP-induced tumorigenesis in mice.

Biochemical characterization of a mitomycin C-resistant human bladder cancer cell line

This study describes characteristics of a mitomycin C (MMC)‐resistant human bladder cancer cell line, J82/MMC‐2, which was established by repeated in vitro exposures of a 6‐fold MMC‐resistant variant (J82/MMC) to 18 nM MMC. A 9.6‐fold higher concentration of MMC was required to kill 50% of the J82/MMC‐2 sub‐line compared with parental cells (J82/WT). NADPH cytochrome P450 reductase and DT‐diaphorase activities were significantly lower in J82/MMC‐2 cells compared with J82/WT, suggesting that reduced sensitivity of J82/MMC‐2 cells to MMC resulted from impaired drug activation. Consistent with this hypothesis, the formation of MMC‐alkylating metabolites was significantly lower in J82/MMC‐2 cells compared with J82/WT. Furthermore, DT‐diaphorase activity in J82/MMC‐2 cells was significantly lower compared with the 6‐fold MMC‐resistant variant. Glutathione (GSH) levels were comparable in all 3 cell lines. Although GSH transferase (GST) activity was significantly higher in the J82/MMC‐2 cells compared with J82/WT, this enzyme activity did not differ between 6‐ and 9.6‐fold MMC‐resistant variants. Whereas DNA polymerase α mRNA expression was comparable in these cell lines, levels of DNA ligase I mRNA were slightly lower in both MMC‐resistant variants relative to J82/WT. However, the DNA polymerase β mRNA level was markedly higher in the J82/MMC‐2 cell line compared with either J82/WT or J82/MMC. Thus, emergence of a higher level of resistance to MMC in J82/MMC‐2 cells compared with J82/MMC may be attributed to (i) impaired drug activation through further reduction in DT‐diaphorase activity and (ii) enhanced DNA repair through over‐expression of DNA polymerase β.

Characterization of a human bladder cancer cell line selected for resistance to BMY 25067, a novel analogue of mitomycin C

This study describes characteristics of a human bladder cancer cell line, SCaBER/R, selected for resistance to a mitomycin C (MMC) analogue BMY 25067. The SCaBER/R cell line was isolated by repeated 24 h exposures of the parental cells to 0.09 microM BMY 25067 (IC90, 24 h drug exposure) over a period of about 180 days. Approximately 2.2-fold higher concentration of BMY 25067 was required to kill 50% of the SCaBER/R cell line compared with parental cells (p < 0.001). The IC20 and IC90 values for BMY 25067 were also significantly higher in the SCaBER/R cell line than in SCaBER. Unlike most MMC resistant cell lines, the SCaBER/R cell line displayed a marked cross-resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and lacked cross-resistance to cisplatin, doxorubicin or VP-16. The SCaBER/R cell line also displayed a marked cross-resistance to the parent drug (MMC) and BMY 25282, another analogue of MMC. NADPH cytochrome P450 reductase activity, an enzyme implicated in bio-reductive activation of MMC, did not differ significantly in these cells. DT-diaphorase activity, another MMC activation enzyme, was significantly lower in the SCaBER/R cell line when compared to the SCaBER cells. These results suggest that relatively lower sensitivity of SCaBER/R cell line to MMC and BMY 25067 may result from impaired drug activation. Cellular levels of glutathione (GSH) and GSH-transferase (GST), which have been suggested to affect the cytotoxicity of MMC, were comparable in SCaBER and SCaBER/R cell lines. BMY 25067 induced DNA interstrand cross-links (DNA-ISC) could not be detected in either of the cell lines even at drug concentrations which produced a significant cell kill. These findings suggest that (a) cellular resistance to BMY 25067 in the SCaBER/R cell line may be due to impaired drug activation, and (b) the nature of the cytotoxic produced by BMY 25067 may be different from that of MMC.

Mechanism of increased sensitivity to etoposide in a mitomycin C‐resisitant human bladder cancer cell line

The mechanism of increased sensitivity to etoposide (VP‐16) in a human bladder cancer cell line (J82/MMC‐2), which is >9‐fold more resistant to mitomycin C (MMC) compared with parental cells (J82/WT), was investigated. Colony formation assays, following 1 hr drug exposure, revealed that about a 2.2‐fold higher concentration of VP‐16 was required to kill 50% of the J82/WT cell line compared with J82/MMC‐2. The MTT assays, following continuous drug exposure, also showed that the J82/MMC‐2 cell line was significantly more sensitive to VP‐16 compared with J82/WT. Accumulation of VP‐16 was significantly higher in the J82/MMC‐2 cell line compared with J82/WT at every drug concentration tested. Likewise, intracellular VP‐16 retention was significantly higher in the J82/MMC‐2 cell line compared with J82/WT when drug uptake was measured as a function of varying incubation time and at a fixed VP‐16 concentration. The efflux of VP‐16 from the J82/MMC‐2 cell line was equivalent to that from J82/WT. In agreement with the results of drug uptake studies, the levels of VP‐16‐induced protein‐DNA complexes were markedly higher in the J82/MMC‐2 cell line compared with J82/WT. The catalytic activity of topoisomerase II (topo II) in 0.35 M NaCl nuclear extract of J82/WT cells was equivalent to that of J82/MMC‐2. The levels of topo II mRNA were also comparable in these cells. Our results suggest that the mechanism responsible for the collateral sensitivity of the J82/MMC‐2 cell line to VP‐16 may be attributable to a relatively higher drug accumulation in this cell line compared with parental cells. Int. J. Cancer 70:612–618.

Biochemical mechanism of cross-resistance to paclitaxel in a mitomycin c-resistant human bladder cancer cell line

9-fold more resistant to mitomycin C (MMC) than parental cells (J82/WT). The IC(50) values for paclitaxel in J82/WT and J82/MMC-2 cell lines were 0.7+/-0.03 and 2.8+/-0.7 microM, respectively (P<0. 05). Thus, the J82/MMC-2 cell line exhibited 4-fold cross-resistance to paclitaxel compared with J82/WT. Intracellular accumulation of [(3)H]paclitaxel was comparable in J82/WT and J82/MMC-2 cell lines. There were no qualitative or quantitative differences between the J82/WT and J82/MMC-2 cell lines in terms of their alpha-tubulin and beta-tubulin contents. Paclitaxel-induced apoptosis could not be detected in either cell line over a wide range of drug concentrations. These results indicate that cross-resistance to paclitaxel in the J82/MMC-2 cell line is not linked to reduced drug accumulation, increased drug efflux, alterations in tubulin content or reduced paclitaxel-induced apoptosis. Paclitaxel-induced DNA strand breakage, however, determined by alkaline elution, was markedly lower in the J82/MMC-2 cell line than in J82/WT. These results suggest that paclitaxel cross-resistance in J82/MMC-2 may be attributed to reduced paclitaxel-induced DNA strand breakage. The precise mechanism of reduced paclitaxel-induced DNA strand breakage in J82/MMC-2 cell line relative to J82/WT cells, however, remains to be elucidated.