Absorption and chemopreventive targets of sulforaphane in humans following consumption of broccoli sprouts or a myrosinase-treated broccoli sprout extract.

SCOPE: Sulforaphane (SFN), an isothiocyanate derived from crucifers, has numerous health benefits. SFN bioavailability from dietary sources is a critical determinant of its efficacy in humans. A key factor in SFN absorption is the release of SFN from its glucosinolate precursor, glucoraphanin, by myrosinase. Dietary supplements are used in clinical trials to deliver consistent SFN doses, but myrosinase is often inactivated in available supplements. We evaluated SFN absorption from a myrosinase-treated broccoli sprout extract (BSE) and are the first to report effects of twice daily, oral dosing on SFN exposure in healthy adults. METHODS AND RESULTS: Subjects consumed fresh broccoli sprouts or the BSE, each providing 200 µmol SFN daily, as a single dose and as two 100-µmol doses taken 12 h apart. Using HPLC-MS/MS, we detected ∼3 x higher SFN metabolite levels in plasma and urine of sprout consumers, indicating enhanced SFN absorption from sprouts. Twelve-hour dosing retained higher plasma SFN metabolite levels at later time points than 24-hour dosing. No dose responses were observed for molecular targets of SFN (i.e. heme oxygenase-1, histone deacetylase activity, p21). CONCLUSION: We conclude that the dietary form and dosing schedule of SFN may impact SFN absorption and efficacy in human trials.

Differential modulation of dibenzo[def,p]chrysene transplacental carcinogenesis: maternal diets rich in indole-3-carbinol versus sulforaphane.

Cruciferous vegetable components have been documented to exhibit anticancer properties. Targets of action span multiple mechanisms deregulated during cancer progression, ranging from altered carcinogen metabolism to the restoration of epigenetic machinery. Furthermore, the developing fetus is highly susceptible to changes in nutritional status and to environmental toxicants. Thus, we have exploited a mouse model of transplacental carcinogenesis to assess the impact of maternal dietary supplementation on cancer risk in offspring. In this study, transplacental and lactational exposure to a maternal dose of 15mg/Kg B.W. of dibenzo[def,p]chrysene (DBC) resulted in significant morbidity of offspring due to an aggressive T-cell lymphoblastic lymphoma. As in previous studies, indole-3-carbinol (I3C, feed to the dam at 100, 500 or 1000ppm), derived from cruciferous vegetables, dose-dependently reduced lung tumor multiplicity and also increased offspring survival. Brussels sprout and broccoli sprout powders, selected for their relative abundance of I3C and the bioactive component sulforaphane (SFN), respectively, surprisingly enhanced DBC-induced morbidity and tumorigenesis when incorporated into the maternal diet at 10% wt/wt. Purified SFN, incorporated in the maternal diet at 400ppm, also decreased the latency of DBC-dependent morbidity. Interestingly, I3C abrogated the effect of SFN when the two purified compounds were administered in equimolar combination (500ppm I3C and 600ppm SFN). SFN metabolites measured in the plasma of neonates positively correlated with exposure levels via the maternal diet but not with offspring mortality. These findings provide justification for further study of the safety and bioactivity of cruciferous vegetable phytochemicals at supplemental concentrations during the perinatal period.

Nonlinear cancer response at ultralow dose: a 40800-animal ED(001) tumor and biomarker study.

Assessment of human cancer risk from animal carcinogen studies is severely limited by inadequate experimental data at environmentally relevant exposures and by procedures requiring modeled extrapolations many orders of magnitude below observable data. We used rainbow trout, an animal model well-suited to ultralow-dose carcinogenesis research, to explore dose-response down to a targeted 10 excess liver tumors per 10000 animals (ED(001)). A total of 40800 trout were fed 0-225 ppm dibenzo[a,l]pyrene (DBP) for 4 weeks, sampled for biomarker analyses, and returned to control diet for 9 months prior to gross and histologic examination. Suspect tumors were confirmed by pathology, and resulting incidences were modeled and compared to the default EPA LED(10) linear extrapolation method. The study provided observed incidence data down to two above-background liver tumors per 10000 animals at the lowest dose (that is, an unmodeled ED(0002) measurement). Among nine statistical models explored, three were determined to fit the liver data well-linear probit, quadratic logit, and Ryzin-Rai. None of these fitted models is compatible with the LED(10) default assumption, and all fell increasingly below the default extrapolation with decreasing DBP dose. Low-dose tumor response was also not predictable from hepatic DBP-DNA adduct biomarkers, which accumulated as a power function of dose (adducts = 100 x DBP(1.31)). Two-order extrapolations below the modeled tumor data predicted DBP doses producing one excess cancer per million individuals (ED(10)(-6)) that were 500-1500-fold higher than that predicted by the five-order LED(10) extrapolation. These results are considered specific to the animal model, carcinogen, and protocol used. They provide the first experimental estimation in any model of the degree of conservatism that may exist for the EPA default linear assumption for a genotoxic carcinogen.

Lymphoma and lung cancer in offspring born to pregnant mice dosed with dibenzo[a,l]pyrene: the importance of in utero vs. lactational exposure.

The fetus and neonate cannot be viewed as "little adults"; they are highly sensitive to toxicity from environmental chemicals. This phenomenon contributes to the fetal basis of adult disease. One example is transplacental carcinogenesis. Animal models demonstrate that environmental chemicals, to which pregnant women are daily exposed, can increase susceptibility of the offspring to cancer. It is uncertain to what degree in utero vs. lactational exposure contributes to cancer, especially for hydrophobic chemicals such as polyhalogenated biphenyls, ethers, dioxins, furans, etc., which can partition into breast milk. We developed a pregnant mouse model in which exposure to the polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), during late gestation, produces an aggressive T-cell lymphoma in offspring between 3 and 6 months of age. Survivors exhibit multiple lung and liver (males) tumors. Here, we adopt a cross-foster design with litters born to dams treated with DBP exchanged with those born to dams treated with vehicle. Exposure to DBP in utero (about 2 days) produced significantly greater mortality than residual DBP exposure only through breast milk (3 weeks of lactation). As previously observed pups in all groups with an ahr(b-1/d) ("responsive") genotype were more susceptible to lymphoma mortality than ahr(d/d) ("non-responsive") siblings. At termination of the study at 10 months, mice exposed in utero also had greater lung tumor multiplicity than mice exposed only during lactation. Our results demonstrate that short exposure to DBP during late gestation presents a greater risk to offspring than exposure to this very hydrophobic PAH following 3 weeks of nursing.

Chemoprevention of dibenzo[a,l]pyrene transplacental carcinogenesis in mice born to mothers administered green tea: primary role of caffeine.

Our laboratory recently developed a mouse model of transplacental induction of lymphoma, lung and liver cancer by the polycyclic aromatic hydrocarbon, dibenzo[a,l]pyrene (DBP). Pregnant B6129SF1 females, bred to 129S1/SvIm males, were treated on day 17 of gestation with an oral dose of 15 mg/kg DBP. Beginning on day 0 of gestation, dams were given (ad lib) buffered water, 0.5% green tea, 0.5% decaffeinated green tea, caffeine or epigallocatechin-3-gallate (EGCG) (both at equivalent concentrations found in tea). The concentration of the teas (and corresponding caffeine and EGCG) was increased to 1.0% upon entering the second trimester, 1.5% at onset of the third trimester and continued at 1.5% until pups were weaned at 21 days of age. Offspring were raised with normal drinking water and AIN93G diet. Beginning at 2 months of age, offspring experienced significant mortalities due to an aggressive T-cell lymphoma as seen in our previous studies. Ingestion of caffeinated, but not decaffeinated, green tea provided modest but significant protection (P = 0.03) against mortality. Caffeine provided a more robust (P = 0.006) protection, but EGCG was without effect. Offspring also developed DBP-dependent lung adenomas. All treatments significantly reduced lung tumor multiplicity relative to controls (P < 0.02). EGCG was most effective at decreasing tumor burden (P = 0.005) by on average over 40% compared with controls. Induction of Cytochrome P450 (Cyp)1b1 in maternal liver may reduce bioavailability of DBP to the fetus as a mechanism of chemoprevention. This is the first demonstration that maternal ingestion of green tea, during pregnancy and nursing, provides protection against transplacental carcinogenesis.

Protective versus promotional effects of white tea and caffeine on PhIP-induced tumorigenesis and beta-catenin expression in the rat.

A 1 year carcinogenicity bioassay was conducted in rats treated with three short cycles of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)/high-fat (HF) diet, followed by 2% white tea (wt/vol), 0.05% epigallocatechin-3-gallate (EGCG) or 0.065% caffeine as sole source of fluid intake. Thirty-two percent of the PhIP/HF controls survived to 1 year, compared with 50, 48.7 and 18.2% in groups given white tea, EGCG and caffeine, respectively. After 1 year, PhIP/HF controls had tumors in the colon, skin, small intestine, Zymbal's gland, salivary gland and pancreas. For all sites combined, excluding the colon, tumor incidence data were as follows: PhIP/HF 69.5%, PhIP/HF + EGCG 48.7%, PhIP/HF + white tea 46.9% and PhIP/HF + caffeine 13.3%. Unexpectedly, a higher incidence of colon tumors was detected in rats post-treated with white tea (69%) and caffeine (73%) compared with the 42% incidence in PhIP/HF controls. In the colon tumors, beta-catenin mutations were detected at a higher frequency after caffeine posttreatment, and there was a shift toward more tumors harboring substitutions of Gly34 with correspondingly high protein and messenger RNA expression seen for both beta-catenin and c-Myc. c-Myc expression exhibited concordance with tumor promotion, and there was a concomitant increase in cell proliferation versus apoptosis in colonic crypts. A prior report described suppression of PhIP-induced colonic aberrant crypts by the same test agents, but did not incorporate a HF diet. These findings are discussed in the context of epidemiological data which do not support an adverse effect of tea and coffee on colon tumor outcome-indeed, some such studies suggest a protective role for caffeinated beverages.

Fetal mouse Cyp1b1 and transplacental carcinogenesis from maternal exposure to dibenzo(a,l)pyrene.

Dibenzo(a,l)pyrene (DBP) is among the most potent carcinogenic polycyclic aromatic hydrocarbons. Previously, we showed that DBP administration to pregnant mice resulted in high mortality of offspring from an aggressive T-cell lymphoma. All mice that survive to 10 months of age exhibit lung tumors with high multiplicity. Recombinant cytochrome P450 (cyp) 1b1 from mice and the homologue 1B1 in humans exhibit high activity toward the metabolic activation of DBP. Targeted disruption of the cyp1b1 gene protects against most DBP-dependent cancers. Mice heterozygous for the disrupted cyp1b1 allele were used to examine the effect of cyp1b1 gene dosage on DBP transplacental carcinogenesis. Dams were treated with 1 or 15 mg/kg of DBP or 50 mg/kg of benzo(a)pyrene. Cyp1b1-null offspring did not develop lymphoma, whereas wild-type and heterozygous siblings, born to dams given the high dose of DBP, exhibited significant mortalities between 10 and 30 weeks of age. At 10 months, all groups had lung adenomas or carcinomas [9.5%, 40.3%, 25.6%, and 100% incidences for controls, benzo(a)pyrene, 1 and 15 mg/kg DBP, respectively]. Cyp1b1 status did not alter benzo(a)pyrene-dependent carcinogenesis. At 1 mg/kg DBP, cyp1b1 status altered the incidence of lung tumors (19.0, 27.8, and 28.6% for nulls, heterozygous, and wild-type, respectively). At 15 mg/kg, tumor multiplicities in cyp1b1 wild-type (9.3) and heterozygous (9.5) offspring were nearly twice that of cyp1b1-null siblings (5.0). These data confirm that cyp1b1 bioactivation of DBP occurs in fetal target tissues, following transplacental exposure, with the thymus and lung as primary and secondary targets, respectively.

Comparison of white tea, green tea, epigallocatechin-3-gallate, and caffeine as inhibitors of PhIP-induced colonic aberrant crypts.

There is growing interest in the possible health benefits of tea. We reported previously on the inhibition by white tea of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)-induced colonic aberrant crypt foci (ACF) in the rat (4). To distinguish between blocking and suppressing effects, and thus provide mechanistic insights into prevention during the initiation versus post-initiation phases of carcinogenesis, white tea, and green tea were administered at 2% (w/v) as the sole source of drinking fluid either 2 wk before and 2 wk during PhIP dosing (100 mg/kg, every other day by oral gavage), or starting 1 wk after the carcinogen and continued until the study was terminated at 16 wk. In the former protocol, each tea produced marginal inhibition of colonic ACF, despite evidence for changes in several hepatic enzymes involved in heterocyclic amine metabolism. Post-initiation, however, the data were as follows (ACF/colon, mean +/- SE): PhIP/water 12.2 +/- 1.5; PhIP/white tea 5.9 +/- 0.9 (** P < 0.01); PhIP/caffeine 5.9 +/- 1.5 (** P < 0.01); PhIP/EGCG 3.5 +/- 0.8 (***P < 0.001); PhIP/green tea 8.9 +/- 1.2 (P = 0.22, not significant). In the latter study, apoptosis was determined using in situ oligo ligation and cleaved caspase-3 assays, whereas cell proliferation was assessed via bromodeoxyuridine (BrdU) incorporation. No consistent changes were seen in apoptosis assays, but BrdU labeling was as follows (percent of cells positive/colonic crypt, mean +/- SE): PhIP/water 10.4 +/- 0.6; PhIP/white tea 8.6 +/- 0.2 (*P < 0.05); PhIP/EGCG 6.0 +/- 0.85 (** P < 0.01); PhIP/caffeine 8.75 +/- 0.45 (*P < 0.05); PhIP/green tea 9.5 +/- 0.4 (P > 0.05, not significant). The data imply that white tea, caffeine, and EGCG may be most effective post-initiation, via the inhibition of cell proliferation in the colon and through the suppression of early lesions.

Indole-3-carbinol in the maternal diet provides chemoprotection for the fetus against transplacental carcinogenesis by the polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene.

The fetus and neonate are sensitive targets for chemically induced carcinogenesis. Few studies have examined the risk/benefit of chemoprotective phytochemicals, given in the maternal diet, against transplacental carcinogenesis. In this study, B6129 SF1/J (AHR(b-1/d)) and 129Sv/ImJ (AHR(d/d)) mice were cross-bred. The polycyclic aromatic hydrocarbon, dibenzo[a,l]pyrene (DBP), was administered to pregnant mice (15 mg/kg, gavage) on gestation day 17, and 2000 p.p.m. indole-3-carbinol (I3C), a chemoprotective phytochemical from cruciferous vegetables, was fed to half of the mice from gestation day 9 until weaning. Offspring born to dams fed I3C exhibited markedly fewer mortalities (P < 0.0001). Maternal dietary exposure to I3C also significantly lowered lung tumor multiplicity (P = 0.035) in offspring surviving to 10 months of age. The I3C chemoprotection was independent of either maternal or fetal AHR genotype. The bioavailability of DBP to fetal target tissue was demonstrated by assessing DNA covalent adduction with a (33)P-post-labeling assay. The bioavailability of I3C was determined by dosing a subset of pregnant mice with [(14)C]-I3C. Addition of chemoprotective agents to the maternal diet during pregnancy and nursing may be an effective new approach in reducing the incidence of cancers in children and young adults.

C-Terminal truncation of rabbit flavin-containing monooxygenase isoform 2 enhances solubility.

Flavin-containing monooxygenases (FMO) are membrane-associated enzymes contributing to oxidative metabolism of drugs and other chemicals. There are no known structures similar enough to FMO to provide accurate insights into the structural basis for differences in metabolism observed among FMOs. To develop an FMO amenable to crystallization, we introduced mutations into rabbit FMO2 (rF2) to increase solubility, decrease aggregation, and simplify isolation. Alterations included removal of 26 AA (Delta26) from the carboxyl-terminus, His(6)-fusion to the amino-terminus and a double Ser substitution designed to reduce local hydrophobicity. Only Delta26 FMO variants retained normal activity, increased the yield of cytosolic rF2 and decreased protein aggregation. Delta26 constructs increased rF2 in cytosol in low (from 2 to 13%), and high salt (from 24 to 62%) conditions. His-fusion proteins, while active and useful for purification, did not affect solubility. Delta26 variants should prove useful for identifying conditions suitable for production of an FMO crystal.

In utero exposure of mice to dibenzo[a,l]pyrene produces lymphoma in the offspring: role of the aryl hydrocarbon receptor.

Lymphoma and leukemia are the most common cancers in children and young adults; in utero carcinogen exposure may contribute to the etiology of these cancers. A polycyclic aromatic hydrocarbon (PAH), dibenzo[a,l]pyrene (DBP), was given to pregnant mice (15 mg/kg body weight, gavage) on gestation day 17. Significant mortalities in offspring, beginning at 12 weeks of age, were observed due to an aggressive T-cell lymphoblastic lymphoma. Lymphocytes invaded numerous tissues. All mice surviving 10 months, exposed in utero to DBP, exhibited lung tumors; some mice also had liver tumors. To assess the role of the aryl hydrocarbon receptor (AHR) in DBP transplacental cancer, B6129SF1/J (AHR(b-1/d), responsive) mice were crossed with strain 129S1/SvIm (AHR(d/d), nonresponsive) to determine the effect of maternal and fetal AHR status on carcinogenesis. Offspring born to nonresponsive mothers had greater susceptibility to lymphoma, irrespective of offspring phenotype. However, when the mother was responsive, an AHR-responsive phenotype in offspring increased mortality by 2-fold. In DBP-induced lymphomas, no evidence was found for TP53, beta-catenin, or Ki-ras mutations but lung adenomas of mice surviving to 10 months of age had mutations in Ki-ras codons 12 and 13. Lung adenomas exhibited a 50% decrease and a 35-fold increase in expression of Rb and p19/ARF mRNA, respectively. This is the first demonstration that transplacental exposure to an environmental PAH can induce a highly aggressive lymphoma in mice and raises the possibility that PAH exposures to pregnant women could contribute to similar cancers in children and young adults.

Haplotype and functional analysis of four flavin-containing monooxygenase isoform 2 (FMO2) polymorphisms in Hispanics.

OBJECTIVES: Previous work defined two flavin-containing monooxygenase 2 (FMO2) alleles. The major allele, FMO2*2 (g.23,238C>T), encodes truncated inactive protein (p.X472) whereas the minor allele, FMO2*1, present in African- and Hispanic-American populations, encodes active protein (p.Q472). Recently, four common (27 to 51% incidence) FMO2 single nucleotide polymorphisms (SNPs) were detected in African-Americans (N=50); they encode the following protein variants: p.71Ddup, p.V113fs, p.S195L and p.N413 K. Our objectives were to: (1) determine the incidence of these SNPs in 29 Hispanic individuals previously genotyped as g.23,238C (p.Q472) and 124 previously genotyped as homozygous g.23,238 T (p.X472); (2) determine FMO2 haplotypes in this population; and (3) assess the functional impact of SNPs in expressed proteins. METHODS: SNPs were detected via allele-specific oligonucleotide amplification coupled with real-time or electrophoretic product detection, or single strand conformation polymorphism. RESULTS: The g.7,700_7,702dupGAC SNP (p.71Ddup) was absent. The remaining SNPs were present but, except for g.13,732C>T (p.S195L), were less common in the current Hispanic study population versus the previously described African-Americans. Only expressed p.N413 K was as active as p.Q472, as determined by methimazole- and ethylenethiourea-dependent oxidation. Haplotype determination demonstrated that the g.10,951delG (p.V113fs), g.13,732C>T (p.S195L) and g.22,060T>G (p.N413 K) variants segregated with g.23,238C>T (p.X472). CONCLUSIONS: SNPs would not alter FMO2 activity in individuals possessing at least one FMO2*1 allele. It is likely that these SNPs will segregate similarly in African-American populations. Therefore, estimates that 26% of African-Americans and 2-7% of Hispanic-Americans have at least one FMO2*1 allele should closely reflect the percentages producing active FMO2 protein.

Phosphoinositide binding regulates alpha-actinin dynamics: mechanism for modulating cytoskeletal remodeling.

The active association-dissociation of dynamic protein-protein interactions is critical for the ability of the actin cytoskeleton to remodel. To determine the influence of phosphoinositide binding on the dynamic interaction of alpha-actinin with actin filaments and integrin adhesion receptors, fluorescence recovery after photobleaching (FRAP) microscopy was carried out comparing wild-type green fluorescent protein (GFP)-alpha-actinin and a GFP-alpha-actinin mutant with a decreased affinity for phosphoinositides (Fraley, T. S., Tran, T. C., Corgan, A. M., Nash, C. A., Hao, J., Critchley, D. R., and Greenwood, J. A. (2003) J. Biol. Chem. 278, 24039-24045). In fibroblasts, recovery of the mutant alpha-actinin protein was 2.2 times slower than the wild type along actin stress fibers and 1.5 times slower within focal adhesions. FRAP was also measured in U87MG glioblastoma cells, which have higher levels of 3-phosphorylated phosphoinositides. As expected, alpha-actinin turnover for both the stress fiber and focal adhesion populations was faster in U87MG cells compared with fibroblasts with recovery of the mutant protein slower than the wild type along actin stress fibers. To understand the influence of alpha-actinin turnover on the modulation of the actin cytoskeleton, wild-type or mutant alpha-actinin was co-expressed with constitutively active phosphoinositide (PI) 3-kinase. Co-expression with the alpha-actinin mutant inhibited actin reorganization with the appearance of enlarged alpha-actinin containing focal adhesions. These results demonstrate that the binding of phosphoinositides regulates the association-dissociation rate of alpha-actinin with actin filaments and integrin adhesion receptors and that the dynamics of alpha-actinin is important for PI 3-kinase-induced reorganization of the actin cytoskeleton. In conclusion, phosphoinositide regulation of alpha-actinin dynamics modulates the plasticity of the actin cytoskeleton influencing remodeling.

Influence of cultivar, maturity, and sampling on blackberry (Rubus L. Hybrids) anthocyanins, polyphenolics, and antioxidant properties.

Total anthocyanin pigments increased from 74.7 to 317 mg/100 g fresh weight (FW) from underripe to overripe for Marion blackberries and from 69.9 to 164 mg/100 g FW for Evergreen blackberries. Total phenolics did not show a marked change with maturity with values slightly decreasing from underripe to ripe. Antioxidant activities, while increasing with ripening, also did not show the marked change that total anthocyanins exhibited. The impact of variation due to plots, subsampling, sample preparation, and measurement on Marion composition was examined in detail. Plot-to-plot and sample differences were the major contributors to variation, with sample preparation being an important contributor for some parameters. Measurement variation was a relatively small component of the total variation. Total anthocyanins for 11 blackberry cultivars ranged from 131 to 256 mg/100 g FW (mean = 198), total phenolics ranged from 682 to 1056 mg GAE/100 g FW (mean = 900), oxygen radical absorbance capacity ranged from 37.6 to 75.5 micromol TE/g FW (mean = 50.2), and ferric reducing antioxidant power ranged from 63.5 to 91.5 micromol TE/g FW (mean = 77.5).

Differences in FMO2*1 allelic frequency between Hispanics of Puerto Rican and Mexican descent.

A polymorphism for the phase I drug-metabolizing enzyme, flavin-containing monooxygenase isoform 2 (FMO2), encoding either truncated inactive protein, FMO2X472 (FMO2.2A), or full-length active enzyme, FMO2Q472 (FMO2.1), is known and exhibits significant interethnic differences in allelic frequency. FMO2 is the major or sole FMO isoform expressed in the lung of most mammals, including nonhuman primates. To date, FMO2.1 has been found only in African-American and Hispanic populations, rendering individuals with this allele subject to drug metabolism that is potentially different from that of the general population. Approximately 26% of African-Americans (n = 180) possess the FMO2*1 allele. In preliminary studies, we initially estimated that 5% of Hispanics (n = 40) have the FMO2*1 allele, but access to large cohorts of individuals of defined national origin has allowed us to determine the occurrence among Mexican-American and Puerto Rican-American groups. We used allele-specific genotyping to detect FMO2*1 from 632 Hispanic individuals, including 280 individuals of Mexican origin and 327 individuals of Puerto Rican origin. Statistical analysis indicated that results from Mexican (five sample sources) and Puerto Rican (three sample sources) samples were consistent with the hypothesis of homogeneity within each group from different sources. Data were subsequently pooled across sources to test for evidence of a difference in occurrence of FMO2*1 between ethnic groups. There was strong evidence (p = 0.0066) that FMO2*1 is more common among Puerto Ricans (7%) than among individuals of Mexican descent (2%). The overall occurrence of FMO2*1 among Hispanics of all origins is estimated to be between 2 and 7%.

Evaluation of chronic dietary exposure to indole-3-carbinol and absorption-enhanced 3,3'-diindolylmethane in sprague-dawley rats.

Indole-3-carbinol (I3C) and 3,3'-diindolylmethane (DIM) are naturally occurring dietary components found in cruciferous vegetables. In the stomach, I3C forms condensation products including DIM. I3C and DIM are marketed as dietary supplements, but little is known about the safety of long-term exposure. Rats were fed either control diet, 1 or 10x the current human dose of absorption-enhanced DIM or 5-7x the maximal recommended dose of I3C. Experimental diets were fed continuously for 3 or 12 months or 2 months followed by control diet for 1 month. Results at 3 or 12 months were similar in most respects. No significant differences between groups were found in blood chemistry. A general decrease in serum enzyme levels in male rats was observed, perhaps indicative of a protective effect. Males fed I3C exhibited higher serum levels of 25-hydroxy-vitamin D3 (25OH-D3). There were no observable differences grossly or histologically between groups, although a high number of hyaline casts were found throughout the kidneys of all animals. In both sexes total hepatic CYP levels were significantly induced by I3C, but not by either dose of DIM. Induction of CYP1A1 and CYP1A2 in liver and CYP1A1 in colon was detected for both sexes fed I3C and the high dose of DIM. CYP3A2 was induced in females fed I3C or the high dose of DIM; males were induced with I3C, but not DIM. No induction of CYP1B1 in the colon was observed in either sex. Long-term exposure to DIM produced no observable toxicity, and comparison to I3C indicates that DIM is a markedly less efficacious inducer of CYP in the rat at doses relevant to human supplementation.

Effect of xenoestrogen exposure on the expression of cytochrome P450 isoforms in rainbow trout liver.

We studied the estrogenic effects of model chemicals in one-year-old juvenile rainbow trout. Methoxychlor (20 mg/kg), diethylstilbestrol (15 mg/kg), 4-tert-octylphenol (25 and 50 mg/kg), and biochanin A (25 and 50 mg/kg) were injected intraperitoneally on days 1, 4, and 7. Fish were sacrificed on day 9 and examined for multiple biomarkers. All of the test chemicals caused increases in plasma vitellogenin levels, a biomarker of estrogenicity. Treatment with the xenoestrogens decreased hepatic lauric acid hydroxylase activity and, as shown by Western blots, also generally reduced expression of hepatic cytochrome P450s 2K1 (CYP2K1), 2M1 (CYP2M1), and 3A27 (CYP3A27) at the protein level. Both doses of biochanin A also significantly induced P4501A (CYPIA) and greatly increased hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity. These findings suggest that methoxychlor, diethylstilbestrol, 4-tert-octylphenol, and biochanin A were all estrogenic and mimicked 17beta-estradiol (E2) in repressing the expression of cytochrome P450 isoforms (CYP2KI, CYP2M1, and CYP3A27) in the rainbow trout liver. Additionally, biochanin A was found to induce CYPIA in this fish species.

Developmental regulation of flavin-containing monooxygenase form 1 in the liver and kidney of fetal and neonatal rabbits.

Flavin-containing monooxygenases (FMOs) comprise a multi-gene family and catalyze the oxygenation of soft nucleophilic sulfur, nitrogen, phosphorus, and selenium in xenobiotics. Previous studies have demonstrated that FMO is regulated developmentally and by the administration of certain steroid hormones. This study examined the expression of FMO form 1 in the livers and kidneys of fetal and neonatal rabbits, from day 25 of gestation through 3 weeks of age, by assaying FMO1 mRNA and protein levels, as well as catalytic activity. FMO1 mRNA and protein expression and FMO catalytic activity were present in fetal livers at the earliest time point measured (day 25 of gestation), although at levels approximately 10% of that found in adult livers. Hepatic FMO1 mRNA levels increased during and after gestation; levels were not significantly different from those measured in adult male livers. FMO1 protein content and activity rose rapidly after birth to reach 70-80% of adult levels by 3 weeks of age. The expression of FMO1 in fetal and neonatal kidneys was markedly lower than in liver. FMO1 mRNA levels never averaged more than 3.4% of adult male liver levels, but did not differ from adult kidney levels at any of the points measured. Protein levels and enzyme activity rose significantly after birth to approximately 30% of the level in adult kidneys by 3 weeks of age. The early developmental appearance of FMO1 suggests a possible role in the metabolism of xenobiotics through transplacental or lactational exposures.

Identification of active flavin-containing monooxygenase isoform 2 in human lung and characterization of expressed protein.

Full-length human (hFMO2.1) and monkey (mFMO2) flavin-containing monooxygenase proteins, which share 97% sequence identity, were produced by baculovirus-mediated expression in insect cells and assayed for S-oxygenation under conditions known to affect FMO activity. Both enzymes demonstrated maximal activity at pH 9.5; but hFMO2.1 retained significantly more activity than mFMO2 did at pH 9.0 and higher. hFMO2.1 also retained significantly more activity than mFMO2 did in the presence of magnesium and all detergents tested. Although hFMO2.1 had more residual activity after heating at 45 degrees C than mFMO2, under some conditions, both had less than 10% of control activity, whereas expressed rabbit FMO2 retained over 50% activity. Screening for NADPH-oxygenation by hFMO2.1, indicated that substituted thioureas with a small cross-sectional area (2.4-4.3 A) are good substrates, whereas 1,3-diphenylthiourea (11.2 A) was not oxygenated. We confirmed the presence of hFMO2.1 in lung tissue from a heterozygous individual (hFMO2*1/hFMO2*2A) by Western analysis and confirmed activity by S-oxygenation. These microsomes also demonstrated a heat-associated loss of activity similar to expressed hFMO2.1. The heat sensitivity of hFMO2.1 may partially explain why activity in post mortem human lung samples has previously been unreported. Individuals that have the FMO2*1 allele-encoding full-length hFMO2.1 may exhibit altered drug metabolism in the lung.