C. Clifford Conaway

Disposition of glucosinolates and sulforaphane in humans after ingestion of steamed and fresh broccoli.

The cancer-chemopreventive effects of broccoli may be attributed, in part, to isothiocyanates (ITCs), hydrolysis products of glucosinolates. Glucosinolates are hydrolyzed to their respective ITCs by the enzyme myrosinase, which is inactivated by heat. In this study, the metabolic fate of glucosinolates after ingestion of steamed and fresh broccoli was compared in 12 male subjects in a crossover design. During each 48-hour baseline period, no foods containing glucosinolates or ITCs were allowed. The subjects then consumed 200 g of fresh or steamed broccoli; all other dietary sources of ITCs were excluded. Blood and urine samples were collected during the 24-hour period after broccoli consumption. Total ITC equivalents in broccoli and total ITC equivalents in plasma and urine were assayed by high-performance liquid chromatography as the cyclocondensation product of 1,2-benzenedithiol. The content of ITCs in fresh and steamed broccoli after myrosinase treatment was found to be virtually identical (1.1 vs. 1.0 μmol/g wet wt). The average 24-hour urinary excretion of ITC equivalents amounted to 32.3 ± 12.7% and 10.2 ± 5.9% of the amounts ingested for fresh and steamed broccoli, respectively. Approximately 40% of total ITC equivalents in urine, 25.8 ± 13.9 and 6.9 ± 2.5 μmol for fresh and steamed broccoli, respectively, occurred as the N-acetyl-L-cysteine conjugate of sulforaphane (SFN-NAC). Total ITC metabolites in plasma peaked between 0 and 8 hours, whereas urinary excretion of total ITC equivalents and SFN-NAC occurred primarily between 2 and 12 hours. Results of this study indicate that the bioavailability of ITCs from fresh broccoli is approximately three times greater than that from cooked broccoli, in which myrosinase is inactivated. Considering the cancer-chemopreventive potential of ITCs, cooking broccoli may markedly reduce its beneficial effects on health.

Phenethyl Isothiocyanate and Sulforaphane and their N-Acetylcysteine Conjugates Inhibit Malignant Progression of Lung Adenomas Induced by Tobacco Carcinogens in A/J Mice

We have shown previously that naturally occurring isothiocyanates derived from cruciferous vegetables and their N-acetylcysteine conjugates inhibit lung adenoma formation induced by tobacco carcinogens in A/J mice at the post-initiation stage. The tumor-inhibitory activity by these compounds is linked with activation of activator protein and induction of apoptosis in lung tissues, suggesting that these compounds may also inhibit the development of adenomas to adenocarcinomas in lung. In this study, the chemopreventive activity of phenethyl isothiocyanate and sulforaphane and their N-acetylcysteine conjugates during progression of lung adenomas to malignant tumors was investigated in A/J mice. Mice were divided into 14 groups and treated with a mixture of 3 micromol benzo(a)pyrene [B(a)P] and 3 micromol 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) given by gavage once weekly for 8 weeks. Twenty weeks after the beginning of carcinogen administration, a total of 20 mice in the treatment groups were sacrificed with an average yield of 7.3 +/- 4.5 lung adenomas per mouse. The remaining mice in each group were fed diets containing phenethyl isothiocyanate (3 and 1.5 mmol/kg diet), sulforaphane (3 and 1.5 mmol/kg diet), phenethyl isothiocyanate-N-acetylcysteine (8 and 4 mmol/kg diet), sulforaphane-N-acetylcysteine (8 and 4 mmol/kg diet) during weeks 21 to 42. Four mice in each of the high-dose treatment groups were sacrificed during weeks 28 and 36 and the bioassay was terminated during week 42; lung tissues were harvested for histopathologic examination of tumors and for cell proliferation (proliferating cell nuclear antigen) and apoptosis (caspase-3) assays using immunohistochemical staining. At termination, the incidence of adenocarcinoma in the 3 mmol/kg diet phenethyl isothiocyanate group and 8 mmol/kg diet phenethyl isothiocyanate-N-acetylcysteine group was reduced to 19% and 13%, respectively, compared with 42% in the carcinogen-treated control group. At the lower doses, phenethyl isothiocyanate and its N-acetylcysteine conjugate also inhibited the incidences of lung adenocarcinoma, however, the decreases were not statistically significant. The lung tumor incidences in groups treated with sulforaphane-N-acetylcysteine in the diet were also significantly reduced to 11% or 16%. Furthermore, the malignant lung tumor multiplicity was significantly reduced from 1.0 tumor/mouse in the carcinogen-treated control group to 0.3 in the sulforaphane low-dose group, 0.3 and 0.4 in the two sulforaphane-N-acetylcysteine groups, and 0.4 in the phenethyl isothiocyanate high-dose group. The malignant tumor multiplicities in other treatment groups were also reduced (0.5-0.8 tumors/mouse), but not significantly. Unlike lung adenocarcinomas, both incidences and multiplicities of lung adenomas were not much affected by treatment with isothiocyanates or their conjugates. Immunohistochemical examination of the lung tumors from all time points indicated that significant reduction in proliferating cell nuclear antigen and induction of apoptosis (terminal nucleotidyl transferase-mediated nick end labeling and caspase-3) were observed in the isothiocyanate and isothiocyanate-N-acetylcysteine-treated groups that showed inhibition of the development of lung adenocarcinomas. The results of the study provide a basis for future evaluation of the potential of phenethyl isothiocyanate and sulforaphane and their conjugates as chemopreventive agents in smokers and ex-smokers with early lung lesions.

Formaldehyde mechanistic data and risk assessment: Endogenous protection from DNA adduct formation

Exposures of rodents to airborne formaldehyde (FA) produce dose-related toxicity, enhanced cell proliferation and squamous cell carcinomas in the nasal passages. The mechanism of FA-induced tumor formation involves DNA-protein crosslink formation and enhanced cell proliferation secondarily to cytotoxicity. The mucociliary apparatus and glutathione protect against low-dose FA-induced effects. Consequently, the mechanistic information is consistent with a very sublinear dose-response curve for tumor formation. The sublinear dose-response of nasal DNA-protein crosslinks levels in rodents and monkeys has been used in the risk assessment of FA.

Enhanced mutagenicity of 2-nitropropane nitronate with respect to 2-nitropropane — possible involvement of free radical species

The mutagenicities of the neutral and the anionic (nitronate) forms of 2-nitropropane (2-NP) were compared in S. typhimurium strains TA98, TA100 and TA102. The latter is a special strain sensitive to compounds producing oxidative damage to DNA at thymine-adenine base pair loci. Neutral 2-NP was not mutagenic in TA98, and produced significant mutagenic response in TA100 and TA102 only at levels of 55 mumoles/plate. In contrast, 2-NP nitronate was significantly mutagenic in TA98 at 14 mumoles/plate and, in strains TA100 and TA102, at approximately 4 mumoles/plate. Inclusion of S9 slightly increased the mutagenicity of 2-NP nitronate in TA102, but decreased it in TA100. The mutagenic response in TA102, but not in TA100, was inhibited by DMSO, a scavenger of hydroxyl radicals, in a dose-dependent manner. When 2-NP nitronate was incubated with thymidine and horseradish peroxidase-H2O2, the formation of 2-NP dimer (2,3-dimethyl-2,3-dinitrobutane), a condensation product of 2-NP free radicals, was observed. This was accompanied by 2-NP nitronate-dependent oxidation of thymidine to thymidine glycol, thymine glycol, 5-hydroxymethyldeoxyuridine and thymine, indicating that hydroxyl radicals and/or other reactive oxygen species capable of causing thymidine damage are also formed in this reaction. As a working hypothesis, we suggest that the genotoxicity of 2-NP nitronate may be due to the generation of DNA-damaging reactive forms of oxygen or 2-NP free radicals.

Chemopreventive potential of fumaric acid, N-acetylcysteine, N-(4-hydroxyphenyl) retinamide and β-carotene for tobacco-nitrosamine-induced lung tumors in A/J mice1

Four agents, fumaric acid (FA), N-acetylcysteine (NAC), N-(4-hydroxyphenyl) retinamide (4-HPR) and beta-carotene (beta-CT), were evaluated for potential chemopreventive activity using the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumor model in female A/J mice. The agents were evaluated in both 16-week and 52-week bioassays at two dose levels corresponding to 0.8 maximum tolerated dose (MTD) and 0.4 MTD administered throughout the bioassay either in the diet (FA, 160 and 80 mmol/kg diet; NAC, 160 and 80 mmol/kg diet; 4-HPR, 4 and 2 mmol/kg diet) or by subcutaneous injection twice a week (beta-CT, 32 and 16 mg/kg b.w.). Mice were treated with a single i.p. dose of 10 micromol NNK in saline 1 week after administration of test agent. Lung adenomas were evaluated in the 16-week bioassay, whereas both adenomas and adenocarcinomas of the lung were determined in the 52-week bioassay. Both bioassays showed that all four agents did not significantly inhibit the total tumor incidence and multiplicity of the lung. However, the incidence of adenocarcinomas was reduced (P < 0.01) at 52 weeks in NNK groups given either 0.8 MTD NAC or 0.8 MTD beta-CT compared with the NNK control group. The decreases in adenocarcinomas were accompanied by corresponding increases in adenomas in these treatment groups. Thus, this study showed that FA, NAC, 4-HPR and beta-CT did not inhibit the total tumor formation, however, at the higher doses both NAC and beta-CT significantly retarded the malignant progression in the lung of NNK-treated A/J mice.

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.

Vinyl chloride mechanistic data and risk assessment: DNA reactivity and cross-species quantitative risk extrapolation

Vinyl chloride produced several tumor types among species. Angiosarcoma of the liver is found in all tested species, including humans with occupational exposures. Vinyl chloride is biotransformed by CYP2E1 to DNA-reactive chloroethylene oxide producing cyclic etheno adducts, which are mutagenic. The dose-response for angiosarcoma of the liver formation in rodents is supralinear, which is consistent with saturation of metabolic activation, and the tumor rate in humans at occupational exposure levels is similar to that for equivalent exposures in rodents.