Role of cytochrome P450 in DNA damage produced by treatment of colon cells with 1,2-dimethylhydrazine

The study of colon cancer has taken advantage of the development of a model in animals in which tumors in the colon are easily induced by chemical treatment. When 1,2-dimethylhydrazine (DMH) is injected into rats tumor growth is observed in colon in preference to other tissues. This observation led us to investigate the Cytochrome P450 system in colon and its participation in the particular “colon sensitivity” to DMH. It has been established that the Cytochrome P450 system participates in the metabolism of DMH and the methyl carbonium product of Cytochrome P450 activation of DMH is responsible for DNA damage which is considered an initial step to carcinogenesis. The Cytochrome P450 system is a reasonable place to search for an explanation of this organotropic effect of DMH and we feel that the knowledge obtained from this study can take us closer to understanding the development of colonic malignancy. In our study we used a human colon cell line (LS174T) treated with DMH. The Cytochrome P450 system in the cells was manipulated with inducers of different isoforms of Cytochrome P450. The effect of DMH on colon cells was measured by determination of O-6-methylguanine which is a DNA adduct derived from the metabolism of this chemical and is associated with development of tumors. Our results support the hypothesis that Cytochrome P450 plays an important role in the damage to cellular DNA by DMH. This damage is increased after induction of Cytochromes P450 1A1 and 2E1. The effect of inhibition of the methyltransferase and glutathione systems on protection against DMH damage in colon demonstrated the importance of the protective role of the former and the lack of effective protection of the latter system. ^

Investigation of I -compounds (newly discovered indigenous DNA modifications) by different approaches

I-compounds are newly discovered covalent DNA modifications detected by the $\sp{32}$P-postlabeling assay. They are age-dependent, tissue-specific and sex-different. The origin(s), chemistry and function(s) of I-compounds are unknown. The total level of I-compounds in 8-10 month old rat liver is 1 adduct in 10$\sp7$ nucleotides, which is not neglectable. It is proposed that I-compounds may play a role in spontaneous tumorigenesis and aging.^ In the present project, I-compounds were investigated by several different approaches. (1) Dietary modulation of I-compounds. (2) Comparison of I-compounds with persistent carcinogen DNA adducts and 5-methylcytosine. (3) Strain differences of I-compounds in relation to organ site spontaneous tumorigenesis. (4) Effects of nongenotoxic hepatocarcinogenes on I-compounds.^ It was demonstrated that the formation of I-compounds is diet-related. Rats fed natural ingredient diet exhibited more complex I-spot patterns and much higher levels than rats fed purified diet. Variation of major nutrients (carbohydrate, protein and fat) in the diet, produced quantitative differences in I-compounds of rat liver and kidney DNAs. Physiological level of vitamin E in the diet reduced intensity of one I-spot compared with vitamin E deficient diet. However, extremely high level of vitamin E in the diet gave extra spot and enhanced the intensities of some I-spots.^ In regenerating rat liver, I-compounds levels were reduced, as carcinogen DNA adducts, but not 5-methylcytosine, i.e. a normal DNA modification.^ Animals with higher incidences of spontaneous tumor or degenerative diseases tended to have a lower level of I-compounds.^ Choline devoid diet induced a drastic reduction of I-compound level in rat liver compared with choline supplemented diet. I-compound levels were reduced after multi-doses of carbon tetrachloride (CCl$\sb4$) exposure in rats and single dose exposure in mice. An inverse relationship was observed between I-compound level and DNA replication rate. CCl$\sb4$-related DNA adduct was detected in mice liver and intensities of some I-spots were enhanced 24 h after a single dose exposure.^ The mechanisms and explanations of these observations will be discussed. I-compounds are potentially useful indicators in carcinogenesis studies. ^

Therapeutic Targeting of ATP7B in Ovarian Carcinoma.

PURPOSE: Resistance to platinum chemotherapy remains a significant problem in ovarian carcinoma. Here, we examined the biological mechanisms and therapeutic potential of targeting a critical platinum resistance gene, ATP7B, using both in vitro and in vivo models. EXPERIMENTAL DESIGN: Expression of ATP7A and ATP7B was examined in ovarian cancer cell lines by real-time reverse transcription-PCR and Western blot analysis. ATP7A and ATP7B gene silencing was achieved with targeted small interfering RNA (siRNA) and its effects on cell viability and DNA adduct formation were examined. For in vivo therapy experiments, siRNA was incorporated into the neutral nanoliposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC). RESULTS: ATP7A and ATP7B genes were expressed at higher levels in platinum-resistant cells compared with sensitive cells; however, only differences in ATP7B reached statistical significance. ATP7A gene silencing had no significant effect on the sensitivity of resistant cells to cisplatin, but ATP7B silencing resulted in 2.5-fold reduction of cisplatin IC(50) levels and increased DNA adduct formation in cisplatin-resistant cells (A2780-CP20 and RMG2). Cisplatin was found to bind to the NH(2)-terminal copper-binding domain of ATP7B, which might be a contributing factor to cisplatin resistance. For in vivo therapy experiments, ATP7B siRNA was incorporated into DOPC and was highly effective in reducing tumor growth in combination with cisplatin (70-88% reduction in both models compared with controls). This reduction in tumor growth was accompanied by reduced proliferation, increased tumor cell apoptosis, and reduced angiogenesis. CONCLUSION: These data provide a new understanding of cisplatin resistance in cancer cells and may have implications for therapeutic reversal of drug resistance.

Studies of the mechanism of daunorubicin carcinogenesis: Modulation by vitamin E

Daunorubicin (DNR) is an anthracycline antibiotic used as a cancer chemotherapeutic agent. However, it causes mammary adenocarcinomas in female Sprague-Dawley (SD) rats. Vitamin E (E) has been found to reduce DNR carcinogenicity. I investigated the mechanism of DNR carcinogenicity and its interaction with E in SD rats by studying DNR-DNA adduct formation and the influence of E status on DNR clearance and free radical producing and detoxifying enzymes.^ The hypothesis was that DNR exerts its tumorigenic effect via free radicals generated during redox cycling and production of reactive intermediates capable of forming DNA adducts. E was postulated to act as a protective agent through a combination of its antioxidant property, modulation of drug clearance and levels of free radical producing and detoxifying enzymes.^ DNA adduct formation was measured by the nuclease P1 $\sp{32}$P-post labeling assay. In vitro, DNR was activated by rat liver microsomes and either NADPH or cumene hydrogen peroxide (CuOOH). Rat liver DNA incubated with this mixture formed two adducts when the cofactor was NADPH and three adducts when CuOOH was used. In vivo, SD rats were treated with i.v. doses of DNR. No detectable DNR-DNA adducts were formed in liver or mammary DNA in vivo, although there was an intensification of endogenous DNA adducts.^ Groups, 1, 2, 3 and 4 of weanling female SD rats were fed 0, 100, 1,000 and 10,000 mg $\alpha$-tocopheryl acetate/kg diet respectively. A comparison of Groups 1 and 4 showed no effect of E status on clearance of 10 mg tritiated DNR/kg body weight over 72 hours. However, liver cleared DNR at a faster rate than mammary epithelial cells (MEC).^ Xanthine oxidase, which catalyzes DNR redox cycling, was significantly decreased in liver and MEC of rats in group 4 compared to groups 1, 2, and 3. Detoxifying enzymes were not dramatically affected by E supplementation. Quinone reductase in MEC was significantly increased in group 4 compared to other groups. Overall, the liver had higher levels of free radical detoxifying enzymes compared to MEC.^ These data support a role of free radicals in DNR carcinogenicity because (1) endogenous DNA adducts formed due to free radical insult are further intensified by DNR treatment in vivo, (2) MEC, the specific target of DNR carcinogenicity, cannot rapidly clear DNR and have a lower free radical detoxifying capability than liver, (3) E supplementation caused lowering of free radical generating potential via xanthine oxidase, and increased DNR detoxification due to elevation of quinone reductase in MEC. ^

Modulation of cytochrome P450 enzyme activity and PAH -induced skin carcinogenesis by naturally occurring coumarins

The present study was designed to determine the potential anticarcinogenic activity of naturally occurring coumarins and their mechanism of action. The results indicated that several naturally occurring coumarins including bergamottin, coriandrin, imperatorin, isopimpinellin, and ostruthin, to which humans are routinely exposed in the diet, were effective inhibitors and/or inactivators of CYP1A1-mediated ethoxyresorufin-O-dealkylase (EROD) or CYP2B1-mediated pentoxyresorufin-O-dealkylase (PROD) in mouse liver microsomes. In addition, bergamottin and corandrin were also found to be inhibitors of purified human P450 1A1 in vitro. Further studies with coriandrin revealed that this compound was a mechanism-based inactivator of P450 1A1 and covalently bound to the P450 1A1 apoprotein. In cultured mouse keratinocytes, bergamottin and coriandrin effectively inhibited the B(a) P metabolism and significantly decreased covalent binding of B(a) P and DMBA to keratinocyte DNA and anti-diol-epoxide-DNA adducts derived from both B(a) P and DMBA in keratinocytes. The data from in vivo experiments showed that bergamottin and coriandrin were potent inhibitors of covalent binding of B (a) P to epidermal DNA and the formation of (+) anti BPDE-DNA adduct, whereas imperatorin and isopimpinellin were more potent inhibitors of covalent binding of DMBA to epidermal DNA. The ability of coumarins to inhibit covalent binding of B (a) P to DNA in mouse epidermis was positively correlated with their inhibitory effect P450 1A1 in vitro, while the inhibitory effect of coumarins on covalent binding of DMBA to epidermal DNA was positively correlated with their inhibitory effects on P450 2B1 and negatively to their inhibitory activity toward P450 1A1. The data from tumor experiments indicated that bergamottin, ostruthin, and coriandrin inhibited tumor initiation by B (a) P in a two-stage carcinogenesis protocol. Bergamottin was most effective in this regard and produced a dose dependent inhibition of papilloma formation in these experiments. In addition, imperatorin was an effective inhibitor of skin tumorigenesis induced by DMBA in SENCAR mouse skin using both a two-stage and a complete carcinogenesis protocol. At dose levels higher than those effective against DMBA, imperatorin also inhibited tumor initiation by B (a) P. The results to date demonstrate that several naturally occurring coumarins possess the ability to block tumor initiation and tumorigenesis by PAHs such as B (a) P and DMBA through inhibition of the P450s involved in the metabolic activation of these hydrocarbons. A working model for the involvement of specific P450s in the metabolic activation of these two PAHs was proposed. ^