After the Women's Health Initiative found that the risks of hormone therapy outweighed the benefits, a need for alternative drugs to treat menopausal symptoms has emerged. We explored the possibility that botanical agents used in Traditional Chinese Medicine for menopausal symptoms contain ERbeta-selective estrogens. We previously reported that an extract containing 22 herbs, MF101 has ERbeta-selective properties. In this study we isolated liquiritigenin, the most active estrogenic compound from the root of Glycyrrhizae uralensis Fisch, which is one of the plants found in MF101. Liquiritigenin activated multiple ER regulatory elements and native target genes with ERbeta but not ERalpha. The ERbeta-selectivity of liquiritigenin was due to the selective recruitment of the coactivator steroid receptor coactivator-2 to target genes. In a mouse xenograph model, liquiritigenin did not stimulate uterine size or tumorigenesis of MCF-7 breast cancer cells. Our results demonstrate that some plants contain highly selective estrogens for ERbeta.

The phylogeography of the California mountain kingsnake, Lampropeltis zonata, was studied using mitochondrial DNA sequences from specimens belonging to the seven recognized subspecies and collected throughout the range of the species. Maximum parsimony and maximum likelihood methods identified a basal split within L. zonata that corresponds to southern and northern segments of its distribution. The southern clade is composed of populations from southern California (USA) and northern Baja California, Mexico. The northern clade is divided into two subclades, a 'coastal' subclade, consisting of populations from the central coast of California and the southern Sierra Nevada Mountains of eastern California, and a 'northeastern' subclade, mainly comprised of populations north of the San Francisco Bay and from the majority of the Sierra Nevada. We suggest that past inland seaways in southwestern California and the embayment of central California constituted barriers to gene flow that resulted in the two deepest divergences within L. zonata. Throughout its evolutionary history, the northern clade apparently has undergone instances of range contraction, isolation, differentiation, and then expansion and secondary contact. Examination of colour pattern variation in 321 living and preserved specimens indicated that the two main colour pattern characters used to define the subspecies of L. zonata are so variable that they cannot be reliably used to differentiate taxonomic units within this complex, which calls into question the recognition of seven geographical races of this snake.

Estrogens produce biological effects by interacting with two estrogen receptors, ERα and ERβ. Drugs that selectively target ERα or ERβ might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERβ-selective compounds have been identified. One class of ERβ-selective agonists is represented by ERB-041 (WAY-202041) which binds to ERβ much greater than ERα. A second class of ERβ-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERβ. Diarylpropionitrile represents a third class of ERβ-selective compounds because its selectivity is due to a combination of greater binding to ERβ and transcriptional activity. However, it is unclear if these three classes of ERβ-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERβ selectivity and pattern of gene expression of these three classes of ERβ-selective compounds compared to estradiol (E2), which is a non-selective ER agonist. U2OS cells stably transfected with ERα or ERβ were treated with E2 or the ERβ-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERβ-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERβ, which is consistent with the finding that most genes regulated by the ERβ-selective compounds were similar to each other and E2. However, there were some classes of genes differentially regulated by the ERβ agonists and E2. Two ERβ-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERβ. Our gene profiling studies demonstrate that while most of the genes were commonly regulated by ERβ-selective agonists and E2, there were some genes regulated that were distinct from each other and E2, suggesting that different ERβ-selective agonists might produce distinct biological and clinical effects.

Indole-3-carbinol (I3C), a natural component of Brassica vegetables, is a promising cancer preventive agent that can reduce the incidence of tumors in reproductive organs when administered in the diet. Here we report on the metabolic fate of radiolabeled I3C in MCF-7 cells. I3C was surprisingly inert to metabolism by these cells with a half-life in medium of approximately 40 h. [(3)H]I3C levels in media declined at a similar rate whether incubation was with cultured cells or in cell-free medium. Neither [(3)H]I3C nor its modified products accumulated in MCF-7 cells and only low levels of intact I3C were detected in cellular fractions. In contrast, I3C represented over 30% of the radioactivity in media even after 72 h. In cytosolic fractions, the 3-(cystein-S-ylmethyl) and 3-(glutathion-S-ylmethyl) conjugates of [(3)H]I3C were the primary conversion products identified after 16 h, representing approximately 50% and approximately 15% of the radioactivity in these fractions, respectively. The reaction of I3C with thiols appears to be nonenzymatic since the cysteine conjugate is produced when I3C is incubated in cell-free medium containing additional cysteine. Both cellular and extracellular proteins were nonspecifically modified with [(3)H]I3C. In medium, proteins are radiolabeled even in the absence of cells, indicating again that enzymatic activation was not required. I3C was also oxidized to indole-3-carboxaldehyde and indole-3-carboxylic acid in culture medium independent of cells. Unexpectedly, 3,3'-diindolylmethane (DIM), an I3C product with in vitro and in vivo biological activity, was detected in cellular fractions and appeared to accumulate in the nucleus, representing approximately 40% of this fraction after 72 h treatment. These findings suggest that MCF-7 cells do not vigorously metabolize I3C and that the major route of reaction is with cellular thiols such as glutathione and proteins. The accumulation of DIM in the nucleus suggests that this product may have a role in the cellular biological activities of I3C.3

Phylogeographic inference can determine the timing of population divergence, historical demographic processes, patterns of migration, and when extended to multiple species, the history of communities. Single-locus analyses can mislead interpretations of the evolutionary history of taxa and comparative analyses. It is therefore important to revisit previous single-locus phylogeographic studies, particularly those that have been used to propose general patterns for regional biotas and the processes responsible for generating inferred patterns. Here, we employ a multilocus statistical approach to re-examine the phylogeography of Lampropeltis zonata. Using nonparametic and Bayesian species delimitation, we determined that there are two well-supported species within L. zonata. Ecological niche modelling supports the delimitation of these taxa, suggesting that the two species inhabit distinct climatic environments. Gene flow between the two taxa is low and appears to occur unidirectionally. Further, our data suggest that gene flow was mediated by females, a rare pattern in snakes. In contrast to previous analyses, we determined that the divergence between the two lineages occurred in the late Pliocene (c. 2.07 Ma). Spatially and temporally, the divergence of these lineages is associated with the inundation of central California by the Monterey Bay. The effective population sizes of the two species appear to have been unaffected by Pleistocene glaciation. Our increased sampling of loci for L. zonata, combined with previously published multilocus analyses of other sympatric species, suggests that previous conclusions reached by comparative phylogeographic studies conducted within the California Floristic Province should be reassessed.

Benomyl (a non-thio fungicide) inhibits hepatic mitochondrial low-Km aldehyde dehydrogenase (mALDH or ALDH2) in ip-treated mice by 50% (IC50) at 7.0 mg/kg, which is surprisingly the same potency range as that for several dithiocarbamate fungicides (and the related alcohol abuse drug disulfiram) and thiocarbamate herbicides previously known for their alcohol-sensitizing action. The mechanism by which benomyl inhibits mALDH was therefore examined, first by comparing the metabolism of benomyl with the aforementioned mono- and dithiocarbamates and second by evaluating the inhibitory potency of the benomyl metabolites. Benomyl in ip-treated mice is converted, via butyl isocyanate, S-(N-butylcarbamoyl)glutathione, and S-(N-butylcarbamoyl)cysteine, to S-methyl N-butylthiocarbamate (MBT), identified as a transient metabolite in liver. MBT is >10-fold more potent than benomyl or butyl isocyanate as an in vivo mALDH inhibitor and is also more potent than the intermediary S-(N-butylcarbamoyl) conjugates. Benomyl and MBT inhibit mouse hepatic mALDH in vitro with IC50s of 0.77 and 8.7 microM, respectively. The potency of MBT is greatly enhanced by fortification of the mitochondria with NADPH alone or plus microsomes giving IC50s of 0.50 and 0.23 microM, respectively. This activation of MBT is almost completely blocked by the cytochrome P450 inhibitor N-benzylimidazole but not by several other cytochrome P450 inactivators. MBT (probably following bioactivation) inhibits mALDH in vivo with an IC50 of 0.3 mg/kg. Two candidate activation products were synthesized for potency determinations. N-Hydroxy MBT (prepared via the trimethylsilyl derivative) was not detected as an MBT metabolite; its low potency also rules against N-hydroxylation as the activation process. MBT sulfoxide, from oxidation of MBT with magnesium monoperoxyphthalate in water, is one of the most potent inhibitors known for mALDH and yeast ALDH in vitro (IC50 0.08-0.09 microM). These findings are consistent with a six-step bioactivation of benomyl, via the metabolites above and N-butylthiocarbamic acid, with MBT as the penultimate and MBT sulfoxide as the ultimate inhibitor of mALDH.

S-Methylation is a new bioactivation mechanism for metam and metabolites of methyl isothiocyanate and dazomet in mice. These soil fumigants are converted to S-methyl metam [MeNHC(S)SMe] which reaches peak levels in liver, kidney, brain, and blood 10-20 min after intraperitoneal (ip) treatment. The half-life of S-methyl metam administered ip is 8-12 min in each of these tissues. S-Methyl metam-oxon [MeNHC(O)SMe] is also detected as a metabolite of each of these soil fumigants on analysis by gas chromatography/mass spectrometry with chemical ionization. The conversion of methyl isothiocyanate to S-methyl metam and its oxon probably involves conjugation with glutathione, hydrolysis to S-(N-methylthiocarbamoyl)-cysteine, cleavage by cysteine conjugate beta-lyase to release metam, and finally methylation and oxidative desulfuration. Metam and dazomet are converted to S-methyl metam by mouse liver microsomes on fortification with S-adenosylmethionine. Metam, methyl isothiocyanate, dazomet, and three metabolites (metam-oxon [MeNHC(O)SH], MeNHC(S)SMe, and MeNHC-(O)SMe) administered ip to mice at 40 mg/kg inhibit low-Km liver mitochondrial aldehyde dehydrogenase and elevate ethanol-dependent blood and brain acetaldehyde levels. Several fungicides including the dialkyldithiocarbamates as the disulfide (thiram and the related alcohol-abuse drug disulfiram) and metal salts (ziram) also yield S-methyl thiocarbamate metabolites. Eight S-alkyl and S-(chloroallyl) thiocarbamate herbicides (EPTC, molinate, butylate, vernolate, pebulate, diallate, sulfallate, and triallate), but not their S-chlorobenzyl analog (thiobencarb), undergo sequential liberation of the thiocarbamic acid and then S-methylation, forming the S-methyl thiocarbamates which are new metabolites and potential aldehyde dehydrogenase inhibitors. The S-methyl mono- and dithiocarbamate metabolites of these herbicides and fungicides are easily identified by retention time on gas chromatography and by mass spectrometry giving [MH]+ plus [R1R2NCO]+ or [R1R2NCS]+, respectively, as the two major ions.

Indole-3-carbinol (I3C), a naturally occurring component of Brassica vegetables, such as cabbage, broccoli, and Brussels sprouts, induces a G1 cell cycle arrest of human breast cancer cells. Structure-activity relationships of I3C that mediate this anti-proliferative response were investigated using synthetic and natural I3C derivatives that contain substitutions at the indole nitrogen. Nitrogen substitutions included N-alkoxy substituents of one to four carbons in length, which inhibit dehydration and the formation of the reactive indolenine. Analysis of growth and cell cycle arrest of indole-treated human breast cancer cells revealed a striking increase in efficacy of the N-alkoxy I3C derivatives that is significantly enhanced by the presence of increasing carbon lengths of the N-alkoxy substituents. Compared to I3C, the half maximal growth arrest responses occurred at 23-fold lower indole concentration for N-methoxy-I3C, 50-fold lower concentration for N-ethoxy-I3C, 217-fold lower concentration for N-propoxy-I3C, and 470-fold lower concentration for N-butoxy-I3C. At these lower concentrations, each of the N-alkoxy substituted compounds induced the characteristic I3C response in that CDK6 gene expression, CDK6 promoter activity, and CDK2 specific enzymatic activity for its retinoblastoma protein substrate were strongly down-regulated. 3-Methoxymethylindole and 3-ethoxymethylindole were approximately as bioactive as I3C, whereas, both tryptophol and melatonine failed to induce the cell cycle arrest, showing the importance of the C-3 hydroxy methyl substituent on the indole ring. Taken together, our study establishes the first I3C structure activity relationship for cytostatic activities, and implicates I3C-based N-alkoxy derivatives as a novel class of potentially more potent experimental therapeutics for breast cancer.