Allan H. Conney

Estrogenic action of DDT and its analogs

The intraperitoneal injection of 50 mg/kg of purified o,p′-DDT, technical grade DDT, purified methoxychlor, or purified p,p′-DDT increased the uterine wet weight by 49, 43, 37, and 28%, respectively, 6 hours after injection. o,p′-DDD, m,p′-DDD, p,p′-DDD, and p,p′-DDE exhibited little or no activity. The intraperitoneal injection of as little as 5 mg/kg or 1 mg/kg of technical grade DDT or o,p′-DDT, respectively, caused a significant increase in the uterine wet weight in immature female rats. The injection of technical grade DDT or o,p′-DDT into ovariectomized adult rats also increased uterine wet weight, indicating that the effect of the DDT analogs was not mediated through the ovaries. Treatment of immature female rats with a 50 mg/kg injection of technical grade DDT or purified o,p′-DDT caused a severalfold stimulation in the incorporation in vitro of glucose-U-14C into lipid, protein, RNA, and acid-soluble constituents in the uterus 6 hours after the dose of DDT. A smaller stimulatory effect was observed with purified p,p′-DDT. Treatment of rats with technical grade DDT, purified o,p′-DDT, methoxychlor or p,p′-DDT 2 hours before an injection of estradiol-17β-6,7-3H inhibited the uptake of estradiol-17β-6,7-3H by the uterus in vivo, possibly by competing for sites that bind estradiol-17β in the uterus. o,p′-DDD, p,p′-DDD, m,p′-DDD and p,p′-DDE did not inhibit the uptake of estradiol-17β-6,7-3H by the uterus. Pretreatment of rats with carbon tetrachloride inhibited the uterotropic action of o,p′-DDT and technical grade DDT. This suggests the possibility that the action of these substances on the uterus may depend on the conversion of the analogs of DDT to estrogenic metabolites.

Inhibition of 7,12-Dimethylbenz(a)anthracene-Induced Skin Tumorigenesis in C57BL/6 Mice by Sulforaphane Is Mediated by Nuclear Factor E2–Related Factor 2

Sulforaphane, a dietary isothiocyanate, possesses potent chemopreventive effects through the induction of cellular detoxifying/antioxidant enzymes via the transcription factor nuclear factor E2-related factor 2 (Nrf2). To investigate carcinogenesis mechanisms related to the regulation of Nrf2, we examined the tumor incidence and tumor numbers per mouse in Nrf2 wild-type (+/+) and Nrf2 knockout (-/-) mice. 7,12-Dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate treatments resulted in an increase in the incidence of skin tumors and tumor numbers per mouse in both genotypes; however, both indices were markedly higher in Nrf2(-/-) mice as compared with Nrf2(+/+) mice. Western blot analysis revealed that Nrf2 as well as heme oxygenase-1, a protein regulated by Nrf2 were not expressed in skin tumors from mice of either genotype, whereas expression of heme oxygenase-1 in Nrf2(+/+) mice was much higher than that in Nrf2(-/-) mice in nontumor skin samples. Next, we examined the chemopreventive efficacy of sulforaphane in mice with both genotypes. Topical application of 100 nmol of sulforaphane once a day for 14 days prior to 7,12-dimethylbenz(a)anthracene/12-O-tetradecanoylphorbol-13-acetate applications decreased the incidence of skin tumor in the Nrf2(+/+) mice when compared with the vehicle-treated group. Importantly, there was no chemoprotective effect elicited by sulforaphane pretreatment in the Nrf2(-/-) mice group. Taken together, our results show for the first time that Nrf2(-/-) mice are more susceptible to skin tumorigenesis and that the chemopreventive effects of sulforaphane are mediated, at least in part, through Nrf2.

Topical applications of caffeine or (−)-epigallocatechin gallate (EGCG) inhibit carcinogenesis and selectively increase apoptosis in UVB-induced skin tumors in mice

SKH-1 hairless mice were irradiated with ultraviolet B (UVB) twice weekly for 20 weeks. These tumor-free mice, which had a high risk of developing skin tumors during the next several months, were then treated topically with caffeine (6.2 μmol) or (−)-epigallocatechin gallate (EGCG; 6.5 μmol) once a day 5 days a week for 18 weeks in the absence of further treatment with UVB. Topical applications of caffeine to these mice decreased the number of nonmalignant and malignant skin tumors per mouse by 44% and 72%, respectively. Topical applications of EGCG decreased the number of nonmalignant and malignant tumors per mouse by 55% and 66%, respectively. Immunohistochemical analysis showed that topical applications of caffeine or EGCG increased apoptosis as measured by the number of caspase 3-positive cells in nonmalignant skin tumors by 87% or 72%, respectively, and in squamous cell carcinomas by 92% or 56%, respectively, but there was no effect on apoptosis in nontumor areas of the epidermis. Topical applications of caffeine or EGCG had a small inhibitory effect on proliferation in nonmalignant tumors as measured by BrdUrd labeling (16–22%), and there was also a similar, but nonsignificant, inhibitory effect on proliferation in malignant tumors. The results suggest a need for further studies to determine whether topical applications of caffeine or EGCG can inhibit sunlight-induced skin cancer in humans.

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (−)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (−)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (−)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (−)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ∼30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ∼5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.

Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. I. Determination of substrate specificity by the cytochrome P-450 and P-448 fractions.

The reconstituted liver microsomal hydroxylation system was used to study the formation of a metabolite-cytochrome P-450 complex absorbing maximally at 455 nm, with benzphetamine and N-hydroxyamphetamine as substrates. Complex formation required the presence of NADPH, substrate, NADPH-cytochrome c reductase, lipid, and cytochrome P-450, indicating that metabolism of the substrate is essential. In the presence of fixed amounts of lipid and NADPH-cytochrome c reductase, the rate of complex formation with cytochrome P-450 isolated from phenobarbital-treated rats was much greater than that observed with cytochrome P-48 from 3-methylcholanthrene-treated rats or rabbits. These results are consistent with recent studies indicating that different forms of cytochrome P-450 with distinct spectral, catalytic, and immunological properties exist in liver microsomes.Abstract The reconstituted microsomal hydroxylation enzyme system from rats treated with phenobarbital exhibited high activity for benzphetamine N-demethylation, but very low activity for 3,4-benzpyrene hydroxylation. However, when the cytochrome P-450 fraction from phenobarbital-treated rats was replaced by the cytochrome P-448 fraction from rats treated with 3-methylcholanthrene, the N-demethylation of benz-phetamine was decreased while the hydroxylation of 3,4-benzpyrene was greatly increased. On the other hand, the reconstituted system from 3-methylcholanthrene-treated rats showed good benzpyrene hydroxylase activity which could be greatly decreased if the cytochrome P-448 fraction was replaced by the P-450 fraction from rats treated with phenobarbital. These and other experiments indicate that the substrate specificity of the hydroxylation system resides in the cytochrome fraction rather than in the reductase or lipid fraction, and the data suggest that cytochromes P-450 and P-448 have different catalytic activities.

Cigarette Smoking: Stimulatory Effect on Metabolism of 3,4-Benzpyrene by Enzymes in Human Placenta

The enzymatic hydroxylation of 3,4-benzpyrene was not detected in human placentas obtained after childbirth from nonsmokers, whereas this enzyme activity was present in placentas obtained from individuals who smoked cigarettes. The degree of induction of benzpyrene hydroxylase caused by cigarette smoking varied in different individuals. Treatment of pregnant rats with benzpyrene increased the activity of this hydroxylase in the placenta.

High mutagenicity and toxicity of a diol epoxide derived from benzo[a]pyrene

Abstract (±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BP 7,8-diol-9,10-epoxide) is a suspected metabolite of benzo[a]pyrene that is highly mutagenic and toxic in several strains of Salmonella typhimurium and in cultured Chinese hamster V79 cells. BP 7,8-diol-9,10-epoxide was approximately 5, 10 and 40 times more mutagenic than benzo[a]pyrene 4,5-oxide (BP 4,5-oxide) in strains TA 98 and TA 100 of S. typhimurium and in V79 cells, respectively. Both compounds were equally mutagenic to strain TA 1538 and non-mutagenic to strain TA 1535 of S. typhimurium . The diol epoxide was toxic to the four bacterial strains at 0.5–2.0 nmole/plate, whereas BP 4,5-oxide was nontoxic at these concentrations. In V79 cells, the diol epoxide was about 60-fold more cytotoxic than BP 4,5-oxide.

Inhibitory effect of curcumin and some related dietary compounds on tumor promotion and arachidonic acid metabolism in mouse skin

Topical application of curcumin, the major yellow pigment in turmeric and curry, has a potent inhibitory effect on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced tumor promotion in mouse skin. The structurally related compounds chlorogenic acid, caffeic acid and ferulic acid are less potent inhibitors. Curcumin is a potent inhibitor of TPA-induced ornithine decarboxylase activity and inflammation in mouse skin whereas chlorogenic acid, caffeic acid and ferulic acid are only weakly active or inactive. Curcumin is a potent inhibitor of arachidonic acid-induced inflammation in vivo in mouse skin, and this compound is also a potent inhibitor of epidermal lipoxygenase and cyclooxygenase activity in vitro. Although chlorogenic acid is only weakly active as an inhibitor of epidermal lipoxygenase activity and TPA-induced ear inflammation, it is more active than caffeic acid and ferulic acid. The inhibitory effects of curcumin, chlorogenic acid, caffeic acid and ferulic acid on TPA-induced tumor promotion in mouse skin parallel their inhibitory effects on TPA-induced epidermal inflammation and epidermal lipoxygenase and cyclooxygenase activities. Examination of the structural features of curcumin required for its biological activity indicate that free hydroxyl groups on the benzene rings are not required for inhibition of TPA-induced ornithine decarboxylase activity and inflammation in mouse skin.

Combined Inhibitory Effects of Curcumin and Phenethyl Isothiocyanate on the Growth of Human PC-3 Prostate Xenografts in Immunodeficient Mice

Earlier studies using prostate cancer cells in culture showed that phenethyl isothiocyanate (PEITC) and curcumin have significant chemopreventive and possibly chemotherapeutic effects. However, their in vivo effects are still lacking. Hence, this study was undertaken to determine the possible in vivo efficacy of prostate cancer-prevention as well as cancer-therapeutic treatment by PEITC and curcumin alone or in combination. We evaluated the effects on tumor growth in vivo, using NCr immunodeficient (nu/nu) mice bearing s.c. xenografts of PC-3 human prostate cancer cells. Molecular biomarkers representing proliferation and apoptosis were determined. Continued i.p. injection of curcumin or PEITC (6 and 5 mumol; thrice a week for 28 days), beginning a day before tumor implantation significantly retarded the growth of PC-3 xenografts. Combination of i.p. administration of PEITC (2.5 mumol) and curcumin (3 mumol) showed stronger growth-inhibitory effects. Next, we evaluated the cancer-therapeutic potential of curcumin and PEITC in mice with well-established tumors, and the results showed that PEITC or curcumin alone had little effect, whereas combination of curcumin and PEITC significantly reduced the growth of PC-3 xenografts. Immunohistochemistry staining and Western blot analysis revealed that the inhibition of Akt and nuclear factor-kappaB signaling pathways could contribute to the inhibition of cell proliferation and induction of apoptosis. Taken together, our results show that PEITC and curcumin alone or in combination possess significant cancer-preventive activities in the PC-3 prostate tumor xenografts. Furthermore, we found that combination of PEITC and curcumin could be effective in the cancer-therapeutic treatment of prostate cancers.

Pregnenolone-16α-carbonitrile: A new type of inducer of drug-metabolizing enzymes

Pretreatment of female rats with pregnenolone-16α-carbonitrile (PCN) for 212 days resulted in a significant increase in cytochrome P-450 content and NADPH-cytochrome c reductase activity in hepatic microsomes. The spectral characteristics of the microsomal hemoprotein from PCN-treated rats were similar to those obtained from untreated and phenobarbital (PB)-treated rats but different from 3-methylcholanthrene (3-MC)-treated animals. However, the specificity of the induction effect of PCN on the oxidative metabolism of benzphetamine, ethylmorphine, and 3,4-benzpyrene differed from the specificity of either PB or 3-MC. Studies on the induction of enzymes that metabolize the above three substrates revealed that PB, PCN, and 3-MC exerted their greatest stimulatory effect on benzphetamine N-demethylation, ethylmorphine N-demethylation, and 3,4-benzpyrene hydroxylation, respectively.