Sulforaphane and its metabolite mediate growth arrest and apoptosis in human prostate cancer cells.

The relation between the consumption of cruciferous vegetables and reduced prostate cancer occurrence has been documented, although the responsible phytochemicals are unknown. The effects of sulforaphane (SFN) which occurs as the precursor glucosinolate in broccoli and other cruciferous vegetables, and its metabolite N-acetylcysteine conjugate (SFN-NAC) on prostate cancer cells were investigated. SFN and SFN-NAC were analyzed with the androgen-dependent human prostate cancer LNCaP cell line model. Cell growth and apoptosis were determined with the expression of androgen receptor and prostate specific antigen, DNA synthesis, cell cycle progression, DNA strand breaks and caspase activation to ascertain the effects and mechanism. SFN and SFN-NAC were demonstrated for the first time to mediate a dose-dependent apoptosis and growth arrest in the prostate cancer cells. Caspases were activated and DNA strand breaks were detected in apoptotic cells. The expression of phosphorylated and dephosphorylated androgen receptors, and the production of prostate specific antigen were attenuated. The expression of cyclin D1 and DNA synthesis were inhibited along with G1 cell cycle block, causing decreased cell density and growth. SFN and its metabolite SFN-NAC have similar activities to induce growth arrest and apoptosis, indicating that the effects of SFN are maintained through the metabolic processes. SFN as a dietary component of cruciferous vegetables active in the prevention of prostate cancer is discussed.

Decomposition rates of isothiocyanate conjugates determine their activity as inhibitors of cytochrome p450 enzymes.

Thiol conjugates of isothiocyanates (thiol-ITCs) are metabolites of ITCs formed in the mercapturic acid pathway in mammals. They are effective chemopreventive agents in mouse lung tumor bioassays and in other models. Thiol-ITCs are inhibitors of P450s, but it has not been determined if P450 inhibition is due to conjugates themselves or to parent ITCs released by deconjugation reactions. In studies of mechanism of chemopreventive action of thiol-ITCs, rates of deconjugation of Cys, GSH, and N-acetyl-L-cysteine (NAC) conjugates of benzyl isothiocyanate (BITC), phenethyl isothiocyanate (PEITC), 6-phenylhexyl isothiocyanate (PHITC), and sulforaphane (SFN), expressed as the first-order rate constant k(1) and the half-life of decomposition Dt(1/2), were measured in aqueous solutions at pH 7.4 and 37 degrees. The Dt(1/2)s for the Cys conjugates were severalfold shorter than the Dt(1/2)s for respective GSH conjugates, while the Dt(1/2)s for the NAC conjugates were the longest. Cleavage of thiol conjugates was pH dependent, much slower under acidic conditions than at pH 7.4. Inhibition of P450 enzymes by thiol-ITCs was followed using PROD (pentoxyresorufin O-dealkylation) for P450 2B1 and EROD (ethoxyresorufin O-dealkylation) for P450 1A1. The inhibition of PROD and EROD by aqueous thiol-ITCs increased with preincubation time and was roughly parallel to the extent of decomposition of the conjugate that had occurred, indicating that both potency of the respective parent ITC and the rate of reductive cleavage of the conjugate influenced enzyme inhibition. In the presence of 250-1000 microM GSH, comparable to physiological levels, rates of deconjugation of thiol-ITCs were markedly reduced; inhibition of PROD was also proportionately reduced. Slow rates of decomposition of thiol-ITCs anticipated in plasma and tissues suggests that inhibition of P450 enzymes involved in carcinogen activation by ITCs released from thiol-ITCs may not be a principal mechanism for their tumor inhibitory activity; other mechanisms probably contribute to their chemopreventive activity.

High-performance liquid chromatography-based determination of total isothiocyanate levels in human plasma: application to studies with 2-phenethyl isothiocyanate.

Dietary and pharmacologic isothiocyanates (ITCs) may play a role in reducing the risk of certain cancers. The quantification of ITCs in humans is important both for epidemiological and pharmacokinetic studies. We describe a modification of an HPLC-based assay of urinary ITCs for use with human plasma. The assay utilizes the cyclocondensation reaction of 1,2-benzenedithiol with ITCs present in human plasma, followed by a two-step hexane extraction and analysis by HPLC using UV detection at 365 nm. The method shows linearity and reproducibility with human plasma over a range of 49-3003 nM phenethyl isothiocyanate (PEITC) (r(2) = 0.996 +/- 0.003). A similar degree of linearity was seen with two other biologically occurring conjugates of PEITC: PEITC--N-acetylcysteine (PEITC--NAC) and PEITC--glutathione (PEITC--GSH). The recovery of PEITC assessed on multiple days was 96.6 +/- 1.5% and was 100% for PEITC--GSH and PEITC--NAC. The reproducibility of the assay on multiday samplings showed a mean %CV of 6.5 +/- 0.3% for PEITC, 6.4 +/- 4.3 for PEITC--NAC and 12.3 +/- 3.9 for PEITC--GSH. In clinical studies, mean plasma ITC level of 413 +/- 193 nM PEITC equivalents was determined for a non-dietary-controlled group of 23 subjects. Multiday analysis data from pharmacokinetic plasma sets of 3 subjects taking a single dose of PEITC at 40 mg showed a good CV (range: 16-21%). The applicability of the methodology to pharmacokinetic studies of PEITC in humans is demonstrated.

Modulation of growth of human prostate cancer cells by the N-acetylcysteine conjugate of phenethyl isothiocyanate.

There is growing evidence that thiol conjugates of isothiocyanates present in cruciferous vegetables are effective cancer chemopreventive and potentially active therapeutic agents. The effects of the N-acetylcysteine conjugate of phenethyl isothiocyanate (PEITC-NAC) on tumor cell growth were analyzed in human prostate cancer cell lines LNCaP, androgen-dependent, and DU-145, androgen-independent. Exposure of the cells to PEITC-NAC at high concentrations caused cytolysis, while at lower concentrations PEITC-NAC mediated a dose-dependent growth modulation, with reduction of DNA synthesis and growth rate, inhibition of clonogenicity and induction of apoptosis in both types of prostate cancer cells. PEITC-NAC decreased cells in S and G2M phases of cell cycle, blocking cells entering replicating phases. In parallel, a significant enhancement of cells expressing the cell cycle regulator p21 as well as its intensity was determined using a fluorescent antibody technique. The action of PEITC-NAC was time-dependent, with the magnitude of inhibition increasing to 50-65% after PEITC-NAC exposure for several days. Interaction of tumor cells with dissociation products of PEITC-NAC, PEITC and NAC, are proposed as the mechanism of growth regulation.

Chemoprevention of colonic aberrant crypt foci in Fischer rats by sulforaphane and phenethyl isothiocyanate.

Epidemiological studies have linked consumption of broccoli to a reduced risk of colon cancer in individuals with the glutathione S-transferase M1 (GSTM1) null genotype. GSTs are involved in excretion and elimination of isothiocyanates (ITCs), which are major constituents of broccoli and other cruciferous vegetables and have cancer chemopreventive potential, so it is speculated that ITCs may play a role in protection against human colon cancer. However, there is a lack of data from animal studies to support this. We carried out a bioassay to examine whether sulforaphane (SFN) and phenethyl isothiocyanate (PEITC), major ITCs in broccoli and watercress, respectively, and their corresponding N:-acetylcysteine (NAC) conjugates, show any chemopreventive activity towards azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) in F344 rats. Groups of six male F344 rats were treated with AOM subcutaneously (15 mg/kg body wt) once weekly for 2 weeks. SFN and PEITC and their NAC conjugates were administered by gavage either three times weekly for 8 weeks (5 and 20 micromol, respectively) after AOM dosing (post-initiation stage) or once daily for 3 days (20 and 50 micromol, respectively) before AOM treatment (initiation stage). The bioassay was terminated on week 10 after the second AOM dosing and ACF were quantified. SFN, SFN-NAC, PEITC and PEITC-NAC all significantly reduced the formation of total ACF from 153 to 100-116 (P < 0.01) and multicrypt foci from 52 to 27-38 (more than four crypts/focus; P < 0.05) during the post-initiation treatment. However, only SFN and PEITC were effective during the initiation phase, reducing the total ACF from 153 to 109-115 (P < 0.01) and multicrypt foci from 52 to 35 (more than four crypts/focus; P < 0.05). The NAC conjugates were inactive as anti-initiators against AOM-induced ACF. These findings provide important laboratory evidence for a potential role of SFN and PEITC in the protection against colon cancer.

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 micromol/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 micromol 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.

Disposition and pharmacokinetics of phenethyl isothiocyanate and 6-phenylhexyl isothiocyanate in F344 rats.

Naturally occurring phenethyl isothiocyanate (PEITC) and its synthetic homolog 6-phenylhexyl isothiocyanate (PHITC) are both effective inhibitors of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone-induced lung tumor development in A/J mice and F344 rats. To help explain why PHITC is considerably more efficacious than PEITC in chemopreventive potency, comparative disposition and pharmacokinetics data for male F344 rats were obtained after a single gavage dose of 50 micromol/kg (3.71 microCi/micromol) [14C]PEITC or 50 micromol/kg (6.59 microCi/micromol) [14C]PHITC in corn oil. After [14C]PEITC dosing, whole blood 14C peaked at 2.9 h, with an elimination half-life (T1/2e) of 21.7 h; blood 14C from [14C]PHITC-treated rats peaked at 8.9 h, with an T1/2e of 20.5 h. In lungs, the target organ, the T1/2e for [14C]PHITC and its labeled metabolites were more than twice that for [14C]PEITC and its labeled metabolites. The effective dose (area under the concentration-time curve) for 14C from PHITC was greater than 2.5 times the area under the concentration-time curve of 14C from PEITC in liver, lungs, and several other tissues. During 48 h, approximately 16.5% of the administered dose of [14C]PHITC was expired as [14C]CO2, more than 100 times the [14C]CO2 expired by rats treated with [14C]PEITC. In rats given [14C]PEITC, 88.7 +/- 2.2% and 9.9 +/- 1.9% of the dose appeared in the urine and feces, respectively, during 48 h; however, rats given [14C]PHITC excreted 7.2 +/- 0.8% of the dose of 14C in urine and 47.4 +/- 14.0% in the feces. Higher effective doses of PHITC in the lungs and other organs may be the basis, in part, for its greater potency as a chemopreventive agent.

Evaluation of possible genotoxic mechanisms for acrylonitrile tumorigenicity.

Acrylonitrile (ACN) exposure is associated with tumors in rat brain, Zymbal gland, and mammary gland. Adducts affecting base pairing were formed in isolated DNA exposed in vitro to the ACN metabolite cyanoethylene oxide (CNEO). DNA from liver, which is not a cancer target organ in ACN-exposed rats, contained low levels of 7-(2-oxoethyl)guanine, and adduct believed not to interfere with base pairing. No adducts have been detected in brain DNA from ACN-exposed rats, suggesting that brain tumors may have arisen by mechanisms other than ACN-DNA reactivity. Genotoxicity assays of ACN have indicated no particular carcinogenic mechanism. Positive reverse mutagenesis in Salmonella typhimurium HisG46 base substitution tester strains by ACN is attributable to CNEO. Other in vitro genotoxicity test assays of ACN have yielded mixed results, without consistent effect of metabolic activation. Some positive genotoxicity data for ACN appear to result from artifacts or from non-DNA-reactive mechanisms. In vivo micronucleus, chromosome aberration, and autoradiographic unscheduled DNA synthesis assays were negative for ACN. The comparative genotoxicity of vinyl chloride and ACN indicates that despite other similarities, they cause rodent tumors by different mechanisms. Also, they absence of ACN-DNA adduct formation in the rat brain suggests the operation of epigenetic mechanisms.

Formation of 8-oxodeoxyguanosine in brain DNA of rats exposed to acrylonitrile.

Acrylonitrile (ACN) produces tumors in rats, particularly gliomas of the brain, but tests for genotoxicity have yielded mixed results and no ACN-DNA adducts have been identified in the brain. To examine the possibility that ACN-related brain tumors were not a consequence of binding of ACN to brain DNA, experiments were conducted to investigate possible epigenetic mechanisms. Male Sprague-Dawley rats were exposed to 0, 3, 30, and 300 ppm ACN in drinking water for 21 days, a range that includes doses associated with brain tumorigenesis. In the 30 and 300 ppm ACN groups, 8-oxodeoxyguanosine (8-oxodG) levels were two fold greater than in the controls. Measures of glutathione levels, glutathione peroxidase and catalase were not significantly changed, but cyst(e)ine was somewhat increased. No changes were found in brain cytochrome oxidase activity, which indicates a lack of metabolic hypoxia. Also, no effects on thiobarbituric acid reactive substances were found, indicating a lack of lipid peroxidation. In an additional experiment, male Sprague-Dawley rats were exposed to 0 or 100 ppm ACN in drinking water for 94 days; interim sacrifices were conducted at 3, 10, and 31 days. Levels of brain nuclear DNA 8-oxodG were significantly increased in ACN-exposed rats compared with controls. Another group of animals were given weekly i.v. injections of 5 mg/kg methylnitrosurea and no increases in 8-oxodG were found. These studies suggest the possibility that ACN-induced tumors may be produced by a mode of action involving 8-oxodG. The formation of 8-oxodG is not understood, but does not appear to involve lipid peroxidation or disruption of antioxidant defenses.

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

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.

Inhibition of rat liver cytochrome P450 isozymes by isothiocyanates and their conjugates: a structure-activity relationship study.

A series of arylalkyl and alkyl isothiocyanates, and their glutathione, cysteine, and N-acetylcysteine conjugates were used to study their inhibitory activity toward the dealkylation of ethoxyresorufin (EROD), pentoxyresorufin (PROD), and methoxyresorufin (MROD) in liver microsomes obtained from the 3-methylcholanthrene or phenobarbital-treated rats. These reactions are predominantly mediated by cytochrome P450 (P450) isozymes 1A1 and 1A2, 2B1 and 1A2, respectively. All isothiocyanates inhibited PROD more readily than EROD. Increases in the alkyl chain length of arylalkyl isothiocyanates to C6 resulted in an increased inhibitory potency in these assays; at longer alkyl chain lengths (C8-C10) the inhibitory potency declined. The IC50s for phenethyl isothiocyanate (PEITC) were 47, 46 and 1.8 microM for EROD, MROD and PROD, respectively. Substitution of an additional phenyl group on PEITC also increased the inhibitory potency; the IC50s for 1,2-diphenylethyl isothiocyanate (1,2-DPEITC) and 2,2-diphenylethyl isothiocyanate (2,2-DPEITC) were 0.9 and 0.26 microM for EROD, and 0.045 and 0.13 microM for PROD, respectively. The relative inhibitory potency of PEITC and its conjugates was N-acetylcysteine-PEITC (PEITC-NAC) < glutathione-PEITC (PEITC-GSH) < cysteine-PEITC (PEITC-CYS) < PEITC. The observations that the parent isothiocyanates were more potent inhibitors than the conjugates suggest that dissociation of the conjugate is required for activity. Naturally occurring alkyl isothiocyanates, sulforaphane (SFO) and allyl isothiocyanate (AITC), were very weak inhibitors in the assays. These results suggest the potential of isothiocyanates as structural probes for studying P450 isozymes. In addition, the inhibitory activity of isothiocyanates for PROD correlated with the previously demonstrated tumor inhibitory potency in (4-methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induced A/J mouse lung tumor bioassays, which supports earlier findings that P450 2B1 is one of the major isozymes involved in NNK activation and that inhibition of this isozyme is an important mechanism for the chemopreventive activity of isothiocyanates.

Inhibition of N-nitrosodimethylamine demethylase in rat and human liver microsomes by isothiocyanates and their glutathione, L-cysteine, and N-acetyl-L-cysteine conjugates.

Natural and synthetic isothiocyanates and their conjugates were examined for their inhibitory effects toward rat and human liver microsomal N-dimethylnitrosoamine demethylase (NDMAd) activity using a radiometric NDMAd assay. Substrate concentrations of 30 and 60 microM were used to probe the activity of cytochrome P4502E1 isozyme through the alpha-hydroxylation of NDMA. It was found that alkyl isothiocyanates such as sulforaphane and allyl isothiocyanate displayed very weak inhibition, whereas the arylalkyl isothiocyanates such as benzyl and phenethyl isothiocyanate showed significant inhibition toward rat liver NDMAd activity with IC50 values of 9.0 and 8.3 microM, respectively. More interestingly, glutathione conjugates of benzyl, phenethyl, and 6-phenylhexyl isothiocyanates all inhibited NDMAd at the comparable concentrations. In the phenethyl isothiocyanate conjugates series, there exist marked differences in their inhibitory activity; i.e., its conjugates with L-cysteine (IC50 = 4.3 microM) and with glutathione (IC50 = 4.0 microM) are more potent than its conjugate of N-acetylcysteine (IC50 = 24.0 microM). The same trend was also observed for the human liver microsomal NDMAd activity. The half-lives of these conjugates were determined in the presence of other free thiols from L-cysteine or glutathione using an HPLC system. It was shown that isothiocyanates are released from their conjugates and react with the free thiols present in the solution. The longer half-life of N-acetylcysteine conjugate of phenethyl isothiocyanate as compared to the other conjugates is consistent with its lower inhibitory activity. The inhibition of NDMAd, and therefore cytochrome P4502E1, by isothiocyanate conjugates is most likely due to the action of the free isothiocyanates released from the conjugates. Since cytochrome P4502E1 and other isozymes play important roles in the activation of the tobacco-specific nitrosoamines, these results provide a basis for investigating the potential of isothiocyanate conjugates as chemopreventive agents.

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.

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.

Effects of organic and inorganic selenium compounds on rat mammary tumor cells.

To explore cellular effects of potent organoselenium chemopreventive agents we have used a rat mammary tumor cell line. We demonstrate that 1,4-phenylenebis(methylene) selenocyanate (p-XSC) at a dose of 5 microM is a more potent inhibitor of DNA, RNA and protein synthesis as well as of mitochondrial transmembrane potential than its chemopreventive counterparts benzyl selenocyanate (BSC) and sodium selenite. These differences were also reflected in reduced growth rate by 24 and 48 hr. Cell-cycle and cell-morphology analysis revealed that higher doses of p-XSC (10 microM) caused DNA fragmentation which was accompanied with partial loss of nuclear stainability, whereas BSC caused a noticeable change in cell-cycle distribution and extensive micronucleation. Overall, our results point to cellular targets of selenium compounds which may mediate their chemopreventive activities in mammary tissues.

2-Nitropropane-induced liver DNA and RNA base modifications: differences between Sprague-Dawley rats and New Zealand white rabbits.

2-Nitropropane (2-NP), a hepatocarcinogen in male Sprague-Dawley rats but not, under the same conditions, in male New Zealand White rabbits, induces characteristic base modifications in rat liver DNA and RNA including increases in 8-oxoguanine and the formation of 8-aminoguanine. We compared the levels of these modifications in the two animal species at 6, 18 and 42 h after a single i.p. treatment with 1.12 mmol/kg 2-NP. Significantly less nucleic acid base modifications were found to be produced in rabbit liver than in rat liver. Thus, the relative resistance of the rabbit to the hepatocarcinogenicity of 2-NP correlates with decreased levels of 2-NP-induced liver DNA and RNA base damage.

Subchronic toxicity of benzyl selenocyanate and 1,4-phenylenebis(methylene)selenocyanate in F344 rats.

Chemopreventive agents benzyl selenocyanate (BSC) and 1,4-phenylenebis(methylene)selenocyanate (p-XSC) were fed in NIH-07 diet to male and female F344 rats (4, 2, and 0.5 mg/kg/day for BSC and 20, 10, and 5 mg/kg/day for p-XSC) for 13 weeks. Weight gains were depressed for male and female rats fed 4 and 2 mg/kg/day BSC, females fed 0.5 mg/kg/day BSC, and male rats fed 20 and 10 mg/kg/day p-XSC. At necropsy, no clear treatment-related lesions were noted, but dose-dependent hepatomegaly was observed in both sexes of BSC and p-XSC groups. Plasma transaminases AST and ALT were elevated in the higher dose groups, while hemoglobin, HCT, and RBC were reduced in most BSC and some p-XSC treatment groups. Plasma glucose was reduced in BSC-treated males. Significant histologic findings included moderate to severe hepatic centrilobular hypertrophy with fatty change in all males and females in the 4 mg/kg/day BSC groups and in 9/15 males and 3/15 females in the 2 mg/kg/day BSC groups. Dose-dependent, mild centrilobular hypertrophy with minimal fatty change was observed in the mid- and low-dose BSC groups and in all p-XSC groups. Mild to moderate renal tubular and interstitial nephritis occurred in the 4 mg/kg/day male BSC group. Dietary maximum tolerated dose levels for chemoprevention studies are 0.5 mg/kg/day (3.0 ppm Se) for BSC and 5 mg/kg/day (32.5 ppm Se) for p-XSC, compared to literature values of 2-3 ppm Se for Na2SeO3.

Evaluation of secondary nitroalkanes, their nitronates, primary nitroalkanes, nitrocarbinols, and other aliphatic nitro compounds in the Ames Salmonella assay.

The secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane and nitrocyclopentane, as well as their anionic forms (nitronates); the primary nitroalkanes 1-nitropropane, 1-nitrobutane, and 1-nitropentane and their respective nitronates; the nitrocarbinols 2-nitro-1-propanol, 2-nitro-1-butanol, 3-nitro-2-butanol, and 3-nitro-2-pentanol and their respective nitronates; 2-methyl-2-nitropropane, and 2-nitroso-2-nitropropane were tested in the Ames Salmonella assay using strains TA98, TA100 and TA102. Nitronates of the secondary nitroalkanes 2-nitropropane, 2-nitrobutane, 3-nitropentane, and nitrocyclopentane were significantly mutagenic in Salmonella strains TA100 and TA102 at 10-80 mumoles/plate, but the parent compounds were mutagenic at only a single dose level or were not mutagenic at all in the same dose range. The primary nitroalkanes and the nitrocarbinols were not mutagenic, or only marginally so, at the concentrations tested. The nitronates of the primary nitroalkanes and the nitrocarbinols reprotonated too rapidly under the conditions of the assay for adequate evaluation of mutagenicity. 2-Methyl-2-nitropropane was not mutagenic in strains TA100 and TA102; 2-nitroso-2-nitropropane was also not mutagenic in strains TA100 and TA102, but induced an equivocal mutagenic response in TA98. The positive Salmonella mutation data for the nitronates of the secondary nitroalkanes studied correlate very well with the very slow rate of reprotonation of secondary nitroalkane nitronates at pH 7.7 (Conaway et al. (1991) Cancer Res., 51, 3143), and provide further evidence that nitronates of secondary nitroalkanes, rather than the neutral parent forms with which they may be in equilibrium, are the more proximate mutagenic species.

Comparison of oxidative damage to rat liver DNA and RNA by primary nitroalkanes, secondary nitroalkanes, cyclopentanone oxime, and related compounds.

The hepatocarcinogen 2-nitropropane causes oxidative damage to liver DNA and RNA after administration to rats; increases in 8-hydroxydeoxyguanosine and formation of an unknown moiety (DX1) in DNA, plus increases in 8-hydroxyguanosine and the appearance of two unidentified peaks (RX1 and RX2) in RNA were observed by high-performance liquid chromatography of nucleosides from 2-nitropropane-treated rats using electrochemical detection (E. S. Fiala et al, Cancer Res., 49:5518-5522, 1989). In the present study, damage to Sprague-Dawley rat liver RNA and DNA was assessed to determine whether the characteristic pattern of oxidative nucleic acid damage caused by 2-nitropropane also occurred after i.p. administration of primary nitroalkanes, other secondary nitroalkanes, 2-methyl-2-nitropropane (a tertiary nitroalkane), and cyclopentanone oxime. All of the secondary nitroalkanes and cyclopentanone oxime significantly increased levels of 8-hydroxyguanine in both DNA and RNA and caused the appearance of DX1, RX1 and RX2. The primary nitroalkanes and the tertiary nitroalkane 2-methyl-2-nitropropane did not cause a similar pattern of nucleic acid damage. The rates of reprotonation of nitronates of the secondary nitroalkanes to the respective un-ionized neutral forms at pH 7.7 were more than 20-fold less than the rates of reprotonation of primary nitroalkane nitronates, suggesting that the anionic nitronates, rather than neutral compounds, are more immediately responsible for the DNA and RNA damage observed in vivo. Since 8-hydroxyguanine is a miscoding lesion in DNA, these results suggest the possibility, still to be rigorously tested, that hepatocarcinogenicity may be associated not only with 2-nitropropane but also with other secondary nitroalkanes as well as with those ketoximes that are capable of being converted to secondary nitroalkanes in vivo.