Theresa J. Smith

Mechanisms of Inhibition of Chemical Toxicity and Carcinogenesis by Diallyl Sulfide (DAS) and Related Compounds from Garlic

Diallyl sulfide (DAS) is a flavor compound derived from garlic and is sequentially converted to diallyl sulfoxide (DASO) and diallyl sulfone (DASO(2)) by cytochrome P(450) 2E1 (CYP2E1). These compounds have been shown to reduce the incidence of a multitude of chemically induced tumors in animal models. The impediment of phase I activation of these carcinogens is hypothesized to be accountable for the reduction in tumor incidence. Indeed, DAS, DASO and DASO(2) are competitive inhibitors of CYP2E1. DASO(2), in addition, is a suicide inhibitor of CYP2E1. These compounds have been shown to reduce carbon tetrachloride-, N-nitrosodimethylamine- and acetaminophen-induced toxicity in rodents. All three chemicals are substrates for CYP2E1. The protective effect was observed when the organosulfur compounds were given before, during or soon after chemical treatment. DAS and DASO(2) inhibited the bioactivation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and related lung tumorigenesis in A/J mice. Because CYP2E1 does not play a key role in NNK activation, the inhibition of other CYP enzymes active in NNK metabolism is likely. DAS also has been shown to induce other CYP and phase II enzymes as well as decrease hepatic catalase activity. All of these effects are observed at concentrations much higher than what is normally ingested by humans. The biological activities of garlic and its related compounds at lower concentrations that mimic human consumption remain to be studied further.

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by inducible and constitutive cytochrome P450 enzymes in rats☆

The tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces tumor formation in the liver, lung, nasal cavity, and pancreas of rats. Metabolic activation is required for the tumorigenicity of this compound. The involvement of cytochrome P450 enzymes in NNK bioactivation was investigated in rats by studies with chemical inducers and antibodies against P450s. Liver microsomal enzymes catalyzed the formation of 4-oxo-1-(3-pyridyl)-1-butanone (keto aldehyde), 4-hydroxy-1-(3-pyridyl)-1-butanone (keto alcohol), 4-(methylnitrosamino)-1-(3-pyridyl-N-oxide)-1-butanone (NNK-N-oxide), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) from NNK. When the activity was expressed on a per nanomole P450 basis, treatments of rats with 3-methylcholanthrene (MC), phenobarbital (PB), pregnenolone 16-alpha-carbonitrile (PCN), Aroclor 1254 (AR), safrole (SA), and isosafrole (ISA) increased the keto aldehyde formation in liver microsomes 2.0-, 2.4-, 3.8-, 2.5-, 2.1-, and 1.8-fold, respectively; PB, AR, SA, and ISA increased the keto alcohol formation 1.7-, 1.3-, 2.0-, and 1.3-fold, respectively. The extents of induction were more pronounced when expressed on a per milligram protein basis, due to the higher microsomal P450 contents in the induced microsomes. The formation of NNK-N-oxide was markedly increased by PB and PCN and slightly increased by AR, SA, and ISA. However, the formation of NNAL, the major metabolite due to carbonyl reduction, was not increased by the treatments but was decreased by AR, ISA, and acetone (AC). The kinetic parameters of NNK metabolism by control, MC-, PB-, and PCN-induced liver microsomes were obtained. A panel of monoclonal (anti-1A1, -2B1, -2C11, and -2E1) and polyclonal (anti-1A2, -2A1, and -3A) antibodies were used to assess the involvement of constitutive hepatic P450 enzymes in NNK metabolism. Keto aldehyde formation was inhibited by anti-1A2 and anti-3A (about 15%) but not by others; the formation of keto alcohol was inhibited by anti-1A2, anti-2A1, and anti-3A (by 13-26%). In incubations with lung microsomes, the formation of keto aldehyde, keto alcohol, NNK-N-oxide, and NNAL were observed. With nasal mucosa microsomes, however, only keto aldehyde and keto alcohol formation were appreciable. SA and AC significantly decreased NNK metabolism in lung and nasal mucosa microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)

Modulation of the levels of cytochromes P450 in rat liver and lung by dietary lipid

This study was undertaken to investigate the effect of dietary lipid on the regulation of several constitutive P450 isozymes. Male Sprague-Dawley rats with body weights of 130-140 g were fed either a 20% corn oil (CO) diet or a fat-free (FF) diet for 4 days following 2 days of fasting. Using liver microsomes, the catalytic activities and immunochemically detectable protein levels of P450s 1A1 and 2, 2A1, 2B1 and 2, 2C11, 2E1, and 3A were determined. The microsomes from rats fed the 20% CO diet exhibited 2-fold higher levels in N-nitrosodimethylamine demethylase activity and P450 2E1 protein than those from rats fed the FF diet. The CO group also showed 2.5-fold higher levels in 6 beta-hydroxylation of testosterone and P450 3A protein than the FF group. In contrast, the CO diet did not affect the immunodetectable level of P450 2C11 protein and its catalytic activities such as benzphetamine demethylase activity and 2 alpha-hydroxylation of testosterone. P450 1A1 was not detectable in either group, but 1A2 was 2.5-fold higher in the CO group than in the FF group. In the liver, the P450 2B1 level was very low in both groups as measured by pentoxyresorufin dealkylase activity and the protein level, whereas 2B2 was 2.5-fold higher in the CO diet group. In lung microsomes from rats fed different amounts of CO, an inverse relationship was observed between the P450 2B1 level and the dietary CO level. The results suggest that the constitutive levels of P450 isozymes are modulated by dietary lipid in a selective manner; the levels of hepatic P450s 1A2, 2B2, 2E1, and 3A were regulated positively but the level of pulmonary P450 2B1 was suppressed by dietary lipid.

Metabolism of 4-(methyhiitrosamino)-l-(3-pyridyI)-1-butanone (NNK) a tobacco-specific carcinogen, by rabbit nasal microsomes and cytochrome P450s NMa and NMb

Rabbit nasal olfactory and respiratory microsomes were found to catalyze the alpha-hydroxylation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) with specific activities of 262 and 136 pmol/min/mg protein in the formation of keto aldehyde, and of 318 and 190 pmol/min/mg protein in the formation of keto alcohol respectively. The formation of NNK-N-oxide was observed in experiments with rabbit olfactory and respiratory microsomes, but not with rat nasal microsomes. However, the rat nasal microsomes had higher activity in catalyzing the alpha-hydroxylation of NNK. In a reconstituted system, rabbit P450NMa, a major constitutive P450 isozyme in nasal microsomes, displayed high activities in the formation of the keto aldehyde and the keto alcohol with apparent Km values of 15 and 9 microM respectively. In comparison, rabbit olfactory specific P450NMb had a low activity in catalyzing the formation of keto aldehyde (Km = 186 microM) and no activity in the formation of keto alcohol. The P450NMa-catalyzed oxidation of NNK was inhibited by nicotine and diallyl sulfide. Kinetic studies indicated that nicotine is a competitive inhibitor. These results demonstrate that enzymes in rabbit nasal microsomes, especially P450NMa, efficiently catalyze the bioactivation of NNK.

Chemopreventive potential of thiol conjugates of isothiocyanates for lung cancer and a urinary biomarker of dietary isothiocyanates

Natural and synthetic isothiocyanates (ITCs) are versatile chemopreventive agents in many animal systems. We have shown that phenethyl ITC (PEITC) and 6‐phenylhexyl ITC (PHITC) are potent inhibitors against lung tumorigenesis induced by tobacco nitrosamine 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) in both mouse and rat. The mechanism by which these ITCs inhibited lung tumorigenesis is attributed to their ability to decrease cytochrome P450 (P450) enzyme activities involved in the activation of NNK. Recently, we have found that thiol conjugates of ITCs inhibit P450 enzymes and are effective inhibitors of lung tumorigenesis. This is significant because conjugation with cellular thiols is the major route of ITC metabolism via the mercapturic acid pathway in rodents and humans. The thiol conjugates are less pungent and potentially less toxic, and they are more soluble and chemically less reactive than ITCs. These properties raise the prospect of substituting thiol conjugates for ITCs as chemopreventive agents. Furthermore, although ample rodent studies have established that ITCs inhibit tumorigenesis, the protective role of dietary ITCs against human cancers has not yet been established. As a prerequisite for such human studies, we have developed an HPLC‐based assay, based on the condensation reaction of ITCs or conjugates with 1,2‐benzenedithiol, for measuring a cyclocondensation product in human urine as an uptake biomarker of total ITCs. This assay was validated using urine samples from subjects who had ingested a known amount of watercress or mustard in a controlled diet. The assay is convenient and rapid, showing promise for analyzing urine samples obtained from population‐based studies. Results from two such studies are presented to illustrate the potential application of this biomarker in epidemiologic studies. J. Cell. Biochem. Suppl. 27:76–85.

Mechanisms of Nitrosamine Bioactivation and Carcinogenesis

It was discovered in the mid-1950s by Magee and Barnes that N-nitrosodimethylamine (NDMA) was hepatotoxic. The carcinogenicity of this compound and many other nitrosamines has since been tested by Magee, Druckrey, Preussmann, Schmahl, and others; the results of these pioneer works were summarized in two comprehensive reviews (1,2). The biochemical mechanisms of the carcinogenicity and the structure-activity relationship of the nitrosamines have since been studied by many investigators and the results have been summarized in three recent symposia (3–5). In this chapter, we will discuss the occurrence and carcinogenicity of nitrosamines, the enzyme mechanisms and the organ site of the activation, and the dose-response relationship in carcinogenesis.

Induction of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) activation in rat lung microsomes by chronic ethanol consumption and repeated running exercise.

The effects of both chronic ethanol consumption and repeated exercise on metabolism of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by rat lung microsomes were investigated. Keto aldehyde production was significantly enhanced 52% and 98%, respectively, by the ethanol and exercise, as was keto alcohol production (72% and 76%). 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) production was significantly enhanced only by the exercise (+32%). The combined treatment enhanced the keto alcohol production by 39%, indicating non-additive effects on cytochrome P450 (CYP) isozymes by the different treatments. Immunoblot and metabolic studies revealed an increased activity and content of CYP1A2 and CYP2B induced by the ethanol and exercise treatments as well as an induction of CYP2E1 proteins by the ethanol and combined treatments. These results indicate that both ethanol consumption and running exercise enhance NNK activation by increasing the expression of the CYP enzymes responsible for NNK activation.

Modulation of Xenobiotic Metabolism and Toxicity by Dietary Chemical

Dietary and nutritional factors are known to influence the biotransformation of drugs and environmental chemicals. Early studies by Wattenberg (Wattenberg, 1971) demonstrated that rats on commercial rat chow had a 68-fold higher intestinal benzo(a)pyrene hydroxylase activity than those on a purified diet. In human studies, ingestion of cabbage and brussels sprouts was shown to increase the metabolism of antipyrine and phenacetin (Conney, 1982, Pantuck and Pan tuck, 1978). Human volunteers on a low-protein diet had lower rates of metabolic clearance of antipyrine and theophylline than those on a high-protein diet (Kappas, et al., 1976). Recent studies demonstrated the transcriptional activation of specific cytochrome P450 genes by dietary chemicals (Pan, et al., 1993, Vang, et al., 1990) and the inhibition of nifedipine metabolism in humans after ingestion of grapefruit juice (Bailey, et al, 1990). Many of these studies have been discussed in several recent reviews (Anderson, et al., 1985, Bidlack, et al., 1986, Hathcock, 1985, Yang, et al., 1992, Yang and Yoo, 1988).

Metabolism and activation of nitrosamines catalysed by cytochrome P-450 isoenzymes

Nitrosamines are a group of nitrogenous compounds that are widely distributed in the environment and can also be synthesized endogenously (Bartsch and Montesano, 1984). Many of these compounds are potent carcinogens showing remarkable tissue and species specificity. Since the discovery of the carcinogenicity of NDMA, the metabolism of nitrosamines has been studied extensively (Magee and Barnes, 1967; Lai and Arcos, 1980). Although α-hydroxylation has been shown many years ago to be a key step in the activation of nitrosamines the enzymatic mechanisms of the metabolic activation of some of these compounds are not clearly understood. The lack of such information has hindered our progress in understanding the tissue and species specific carcinogenicity of nitrosamines. In this review, the enzymes and mechanisms involved in the activation of NDMA and other nitrosamines are discussed.