A 28-day gavage toxicity study in Fischer 344 rats with 3-methylfuran.

3-Methylfuran is produced in foods during food processing and preservation techniques that involve heat treatment such as cooking, jarring, canning, and pasteurization. Currently, there are no studies available on the toxicity of 3-methylfuran. We conducted a 28-day gavage toxicity study (7 days per week) using doses of 0.0, 0.1, 0.3, 1.5, 3.0, 6.0, 12.0, and 25.0 mg/kg bw/day in order to determine the dose range needed to establish a no observed adverse effect level and to better characterize nonneoplastic effects including those affecting hematology, clinical biochemistry, gross morphology, and histopathology. Histological changes of the liver were noted in all treated animals and gross changes were noted beginning at 3.0 mg/kg bw/kg. Alterations in the activity of serum enzymes indicative of effects on the liver were observed, including increases in levels of alanine transaminase and alkaline phosphatase at the highest dose. There was a significant increase in serum thyroxine (T4) and triiodothyronine (T3), which was not accompanied by histological changes in the thyroid. For the most part, statistically significant changes were seen only at the highest dose for hematology and at the 2 highest doses for clinical chemistry parameters. In contrast, mild histological lesions in the liver were observed even at the lowest dose of 0.1 mg/kg bw/day.

A 28-day gavage toxicity study in male Fischer 344 rats with 2-methylfuran.

In most thermally treated products, a series of alkylated furan derivatives have been found, in particular 2-substituted alkylfurans such as 2-methylfuran. These methyl analogs are metabolically activated in a similar fashion as the parent furan, yielding highly reactive unsaturated dialdehydes. There is currently limited toxicological data available for 2-methyl furan exposure by any route that makes conducting a risk assessment difficult. In this pilot study, we report the general toxicology findings affecting tissue morphology, histopathology, clinical biochemistry, and hematology in a 28-day gavage study. The liver was the primary target organ that developed dose-dependent toxicity. Relative liver weights were increased by 42% at 25.0 mg/kg/body weight (bw)/day. Histological changes in the liver were observed at 0.4, 1.5, 3.0, 6.0, 12.0, and 25.0 mg/kg bw/day. These changes were not accompanied by clinical changes in the serum enzyme markers such as alanine transaminase, alkaline phosphatase, and aspartate transaminase. Clinical biochemistry markers for kidney were altered, but these were not accompanied by histological changes. The prostate was significantly decreased in size at the 25.0 mg/kg bw/day dose of 2-methyfuran. Some hematological parameters were also altered.

Biomonitoring of human fetal exposure to environmental chemicals in early pregnancy.

The first trimester of human fetal life, a period of extremely rapid development of physiological systems, represents the most rapid growth phase in human life. Interference in the establishment of organ systems may result in abnormal development that may be manifest immediately or programmed for later abnormal function. Exposure to environmental chemicals may be affecting development at these early stages, and yet there is limited knowledge of the quantities and identities of the chemicals to which the fetus is exposed during early pregnancy. Clearly, opportunities for assessing fetal chemical exposure directly are extremely limited. Hence, this review describes indirect means of assessing fetal exposure in early pregnancy to chemicals that are considered disrupters of development. Consideration is given to such matrices as maternal hair, fingernails, urine, saliva, sweat, breast milk, amniotic fluid and blood, and fetal matrices such as cord blood, cord tissue, meconium, placenta, and fetal liver. More than 150 articles that presented data from chemical analysis of human maternal and fetal tissues and fluids were reviewed. Priority was given to articles where chemical analysis was conducted in more than one matrix. Where correlations between maternal and fetal matrices were determined, these articles were included and are highlighted, as these may provide the basis for future investigations of early fetal exposure. The determination of fetal chemical exposure, at the time of rapid human growth and development, will greatly assist regulatory agencies in risk assessments and establishment of advisories for risk management concerning environmental chemicals.

Effects of oral exposure to arsenobetaine during pregnancy and lactation in Sprague-Dawley rats.

Arsenobetaine (ASB) is the major form of arsenic (As) in seafood sources such as molluscs and fish. Limited data demonstrated that ASB toxicity in mammals is minimal; however, data on possible reproductive effects are lacking. This study investigated the tissue distribution and developmental effects of ASB during pregnancy, early postnatal life, and development to adulthood. Pregnant rats were randomly assigned to 3 cohorts and gavaged daily from gestational day 8 (GD8) with ASB in deionized water at 0, 0.1, 1, or 10 mg/kg body weight (bw)/d. Cohort 1 dams were sacrificed on GD20 (n = 6 per dose group), cohort 2 dams and pups were sacrificed on postnatal day 13 (PND13; n = 4 dams per dose group), and cohort 3 pups (n = 2 dams per dose group) were sacrificed on PND90. Residue analysis detected significant levels of ASB in livers of cohort 1 dams and lower levels in cohort 1 GD20 fetuses, as well as in cohort 2 male and female offspring, indicating placental transfer from the maternal circulation in utero. Trace amounts of ASB in dams' milk were found only in the 10-mg/kg bw/d dose cohort 2 (PND13), demonstrating that lactational transfer was limited. ASB levels in liver varied during pregnancy, lactation, and postweaning, with levels falling rapidly as these physiological states progress. Although transfer of ASB through the placenta to the fetuses and to a limited extent through milk was confirmed, ASB exposure during pregnancy and lactation appeared to produce no teratogenic or deleterious effects on reproductive development.

Toxicologic and immunologic effects of perinatal exposure to the brominated diphenyl ether (BDE) mixture DE-71 in the Sprague-Dawley rat.

Brominated diphenyl ethers (BDEs) are persistent environmental contaminants found in human blood, tissues, and milk. To assess the impact of the commercial BDE mixture DE-71 on the developing immune system in relation to hepatic and thyroid changes, adult (F0) rats were exposed to DE-71 by gavage at doses of 0, 0.5, 5, or 25 mg/kg body weight (bw)/d for 21 weeks. F0 rats were bred and exposure continued through gestation, lactation and postweaning. F1 pups were weaned and exposed to DE-71 by gavage from postnatal day (PND) 22 to 42. On PND 42, half of the F1 rats were assessed for toxicologic changes. The remaining F1 rats were challenged with the T-dependent antigen keyhole limpet hemocyanin (KLH) and immune function was assessed on PND 56. Dose-dependent increases in total BDE concentrations were detected in the liver and adipose of all F0 and F1 rats. In F0 rats, increased liver weight, hepatocellular hypertrophy, and decreased serum thyroxine (T4) were characteristic of DE-71 exposure. In F1 rats perinatal DE-71 exposure caused a nondose-dependent increase in body weight and dose-dependent increases in liver weight and hepatocellular hypertrophy. Serum T3 and T4 levels were decreased. In spleen from DE-71 exposed rats the area occupied by B cells declined while the area occupied by T cells increased; however, cellular and humoral immune responses to KLH challenge were not altered. Thus hepatic and thyroid changes in rats exposed perinatally to DE-71 were associated with altered splenic lymphocyte populations, an effect which has been linked to hypothyroidism.

Sex- and age-specific immunomodulatory effects of dietary soya protein isolate and isoflavones in rats.

The present study examined, using rats as a model, the effects of sex and age of exposure to dietary soya components on serum total and soya-specific antibody content. In Expt 1, Sprague-Dawley rats at 50 d of age were fed diets containing 20 % casein or 20 % alcohol-washed soya protein isolate (SPI) with or without supplemental isoflavones (ISF, 250 mg/kg diet) for 70, 190 or 310 d. The offspring were fed the same diets as their parents. In Expt 2, juvenile Sprague-Dawley rats at 30 d of age were fed diets containing 20 % casein with or without supplemental ISF (50 mg/kg diet) or increasing amounts of alcohol-washed SPI (5, 10 or 20 %) for 90 d. Exposure of rats to dietary SPI before the age of 28 d increased serum total IgA and IgM, and induced the production of SPI-specific IgA, IgG, IgM and IgE antibodies. Feeding of juvenile or adult rats with SPI elevated serum total IgA in females, while the opposite occurred in males, and markedly stimulated the production of SPI-specific IgM in females and IgG in males. Our data suggest that the effects of soya proteins and ISF on the production of serum total and SPI-specific antibodies appear to be sex dependent and also related to the age of exposure to soya in rats. However, the physiological significance of these immune responses remains to be determined.

Distribution of isoflavones in samples of serum, liver and mammary glands of rats or pigs fed dietary isoflavones.

BACKGROUND/AIMS: There has been great interest in the potential beneficial and adverse health effects of dietary isoflavones. Determination of tissue concentrations of isoflavone metabolites provides an insight into the potential bioactivity of dietary isoflavones. However, data on the distribution of isoflavones in animal models fed dietary isoflavones are limited. In this study, additional data on the distribution of isoflavones in serum and/or tissues of rats and pigs fed dietary isoflavones were generated. METHODS: Rats (male and female) were fed a casein control diet (containing no isoflavones) and an isoflavone-supplemented diet (containing an alcohol-washed soy protein isolate plus NOVASOY, providing a total of 1,047 mg/kg of total isoflavones). Female pigs were fed a control diet (without soy) containing 17.5 mg/kg of isoflavones, a soy diet containing 582.8 mg/kg of isoflavones or a soy diet supplemented with a daily dose of 2.3 g (equivalent to 42.0 and 14.5 mg/kg of body weight at the onset and end of treatment, respectively) of crystalline genistein. The concentrations of isoflavones in serum and tissues (liver and mammary gland) and in tissues (liver and mammary gland) of pigs were determined via a sensitive and rapid method using liquid chromatography/mass spectrometry. RESULTS: Rats fed the control diet containing no isoflavones had nondetectable levels of isoflavone metabolites in serum, liver and mammary gland samples. Rats fed the isoflavone-supplemented diet had the greatest levels of equol, followed by genistein, daidzein and glycitein, respectively, in their serum, livers and mammary glands. The concentrations of total isoflavones (daidzein, equol and genistein plus glycitein) in serum were significantly (p < 0.05) greater in male rats vs. female rats, but the reverse was true in the case of livers. Concentrations of daidzein, equol, genistein and glycitein were lowest (p < 0.05) in the livers of pigs fed the control diet, and in the mammary glands of female pigs there was only an effect of feeding soy plus genistein on the concentrations of daidzein and equol (p <0.05). CONCLUSIONS: The current data therefore show gender as well as species differences in the tissue distribution of isoflavones.

Evolution of steroid-5alpha-reductases and comparison of their function with 5beta-reductase.

Steroid-5alpha-reductases (SRD5alpha) and steroid-5beta-reductase (SRD5beta) represent a convergence in evolution: they share similar biological functions, but do not have a common ancestor. In vertebrates, SRD5alpha and SRD5beta are involved in C-19 and C-21 steroid biosynthesis, bile acid biosynthesis and erythropoiesis. We compare and contrast the history, evolution, tissue distribution, enzyme characteristics and biological functions of SRD5alpha and SRD5beta and suggest possible future directions for research efforts. Both, the unique and overlapping roles that SRD5alpha and SRD5beta play in steroid hormone metabolism, are indicated. We also present the phylogeny of the SRD5alpha. The main SRD5alpha subfamilies obtained include, not only the well-known SRD5alpha type 1, type 2 and type 3, but also the synaptic glycoprotein (GPSN2)/trans-2,3-enoly-CoA reductase group. Phylogenetic analysis indicated that a eukaryotic ancestor likely underwent duplication events to generate these three subfamilies (type 1/2, type 3 and GPSN2 ancestors); both SRD5alpha type 1/2 and GPSN2 subfamilies may have evolved by ancient duplication events at the early stage of vertebrate and chordate evolution.

Fadrozole and finasteride exposures modulate sex steroid- and thyroid hormone-related gene expression in Silurana (Xenopus) tropicalis early larval development.

Steroidogenic enzymes and their steroid products play critical roles during gonadal differentiation in amphibians; however their roles during embryogenesis remain unclear. The objective of this study was to investigate the expression and activity of aromatase (cyp19; estrogen synthase) and 5 beta-reductase (srd5 beta; 5 beta-dihydrotestosterone synthase) during amphibian embryogenesis. Expression and activity profiles of cyp19 and srd5 beta were first established during Silurana (Xenopus) tropicalis embryogenesis from Nieuwkoop-Faber (NF) stage 2 (2-cell stage; 1h post-fertilization) to NF stage 46 (beginning of feeding; 72 h post-fertilization). Exposures to fadrozole (an aromatase inhibitor; 0.5, 1.0 and 2.0 microM) and finasteride (a putative 5-reductase inhibitor; 25, 50 and 100 microM) were designed to assess the consequences of inhibiting these enzymes on gene expression in early amphibian larval development. Exposed embryos showed changes in both enzyme activities and sex steroid- and thyroid hormone-related gene expression. Fadrozole treatment inhibited cyp19 activity and increased androgen receptor and thyroid hormone receptor (alpha and beta) mRNAs. Finasteride treatment inhibited srd5 beta (activity and mRNA), decreased cyp19 mRNA and activity levels and increased estrogen receptor alpha mRNA. Both treatments altered the expression of deiodinases (thyroid hormone metabolizing enzymes). We conclude that cyp19 and srd5 beta are active in early embryogenesis and larval development in Silurana tropicalis and their inhibition affected transcription of genes associated with the thyroid and reproductive axes.

Oral p-tert-octylphenol exposures induce minimal toxic or estrogenic effects in adult female Sprague-Dawley rats.

Contamination of the environment with endocrine-disrupting chemicals (EDC) has raised concerns about potential health hazards for humans and wildlife. Human and wildlife exposure to one such ubiquitous chemical, p-tert-octylphenol (OP), are likely, due to its persistence in the environment and its presence in food, water, and items of daily use. OP is reported to bind to the estrogen receptor (ER) and alter expression of estrogen-responsive genes. Detrimental effects of OP exposures on the reproductive system have been observed in most, but not all, in vivo experiments. This study examined estrogenic effects of oral exposures of adult female rats to OP. In vitro, OP bound weakly to human ER and a co-activator protein, and accelerated proliferation of MCF-7 cells. Adult Sprague-Dawley rats were given OP by gavage daily for 35 d (25, 50, or 125 mg/kg/d). Body and organ weights and ovarian follicle populations were not significantly altered in OP-exposed adult rats, despite detectable levels of OP in reproductive organs. The estrous cycle of rats was slightly altered, but there were no significant estrogen-like changes in histomorphology or gene expression of the uterus. Prepubertal rats given 125 or 250 mg/kg OP by gavage for 3 d had reduced body weight compared to vehicle-exposed rats but failed to show any uterotrophic response, although 17alpha-ethinyl estradiol (EE, 10 microg/kg/d, ip) induced a threefold increase in uterine weight. Overall, results suggest that toxicity will occur before estrogenic effects with oral exposures to OP. Relevant environmental exposures likely pose little risk for estrogenic effects.

Soy isoflavones modulate azoxymethane-induced rat colon carcinogenesis exposed pre- and postnatally and inhibit growth of DLD-1 human colon adenocarcinoma cells by increasing the expression of estrogen receptor-beta.

We studied the effects of lifetime exposure to dietary soy isoflavones in an azoxymethane (AOM)-induced rat colon cancer model. Male pups born to Sprague-Dawley rats exposed (including during pregnancy and lactation) to soy isoflavones at either no (0 mg = control), low (40 mg), or high (1000 mg) doses/kg diet were weaned and continued receiving their respective parental diets until the end of the study. Weaned rats received 2 subcutaneous injections (15 mg/kg body weight) of AOM 1 wk apart. After 26 wk, rats were killed and the coordinates of colon aberrant crypt foci (ACF) and tumors were determined. Expression of estrogen receptor (ER)-beta was assessed in rat colon tumors and in DLD-1 human colon adenocarcinoma cells exposed to soy isoflavones. Compared with the control, soy isoflavones did not affect incidences or multiplicities of colon ACF or tumors. Low-dose soy isoflavones decreased tumor burden and size compared with the control (P < 0.05). Expression of ERbeta increased in colon tumors of soy isoflavone-treated groups compared with the control. Soy isoflavones dose-dependently arrested the growth of DLD-1 cells and at subcytotoxic levels increased the expression of ERbeta. Our results suggest that pre- and postnatal exposure to dietary soy isoflavones suppresses the growth of colon tumors in male rats. The overexpression of ERbeta in both rat colon tumors and DLD-1 cells caused by soy isoflavones suggests that ERbeta is a critical mediator in mitigating its cancer-preventive effects.

Effects of chronic exposure to octylphenol on the male rat reproductive system.

p-tert-Octylphenol (OP) is a degradation product of alkylphenol ethoxylates. OP is an endocrine disruptor known to bind to the estrogen receptor; however, effects on males are controversial. The objective of this study was to evaluate the effects of chronic exposure to OP on male reproduction. Adult Sprague-Dawley rats were administered OP for 60 d, representing 1.5 cycles of spermatogenesis. Experimental groups included a vehicle control, and three doses of OP (25, 50, or 125 mg/kg body weight [bw]) administered daily by gavage. There was a significant decrease in body weight in the 125-mg/kg group after 60 d of treatment. Both testicular and epididymal weights and histology were not altered by treatment with OP at any of the doses administered. There were no marked differences in cauda epididymal sperm counts at any doses; however, total percent sperm motility was significantly lower in rats exposed to the intermediate dose (50 mg/kg bw). There was an increase in percent static sperm cells in all OP-treated groups, with the intermediate dose (50 mg/kg) displaying a significantly higher proportion of static cells relative to untreated controls. Caput epididymal sperm motility was unaltered by OP treatment. Gene expression profiles of testes from control and high-dose-exposed rats indicate that 14 genes were modulated by at least twofold, although these changes were not statistically significant. Taken together, results from this study indicate that OP treatment of adult rats does not appear to exert major effects on male reproductive endpoints at relevant environmental exposure doses.

An investigation of the effects of methylmercury in rats fed different dietary fats and proteins: testicular steroidogenic enzymes and serum testosterone levels.

Methylmercury (MeHg) is a testicular toxicant causing reduced steroidogenic enzyme activity, reduced serum testosterone (T) and abnormal spermatogenesis in mammals and fowl. It is also known that certain diets can alter androgen metabolism in rats. Previously we have shown that diets used in the current study impact circulating androgen levels and testicular steroidogenic enzyme activities in Sprague Dawley rats in the absence of MeHg. In the present study, we have investigated the impact of imposing an environmental contaminant (MeHg) commonly found in marine mammals and fish onto the rats' dietary intake of different proteins and lipids in order to determine if the different diets could modify MeHg toxicity in rats. Therefore, we examined the effects of MeHg on testicular steroidogenic enzymes and serum testosterone in rats fed diets containing either different protein sources (casein, fishmeal, whey) or different lipid sources (soybean oil, docosahexaenoic acid (DHA), seal oil, fish oil, lard). Male rats 42-45 days of age (18 per group) were assigned to different experimental diets for 28 days after which 6 rats in each group were gavaged daily with 0, 1 or 3 mg/kg body weight (BW)/day MeHg chloride in 5 mM Na(2)CO(3) solution for 14 days while being maintained on their diets. On the 43rd day of dosing, rats were sacrificed and blood plasma and testes frozen (-80 degrees C) until analysis. Microsomal steroidogenic enzyme activities (3beta-HSD, 17-OHase, C-17, 20-lyase, 17beta-HSD) were measured radiometrically. Serum testosterone was determined using ELISA kits. Testis weights were not affected by MeHg. MeHg at 3 mg/kg BW/day caused a reduction (>50%) in the activity of C-17, 20-lyase in all three protein diets and similar reductions in 17-OHase activity were seen in the casein and whey protein fed rats. At 3 mg/kg BW/day, MeHg reduced 17-OHase activity in the DHA diet but had no effect on 3beta-HSD activity and no inhibitory effects on 17beta-HSD activity. MeHg (3 mg/kg BW/day) caused significant reductions in serum T in the whey, soybean oil and fish oil groups. Interestingly, fishmeal protein but not fish oil offered some protection with respect to maintaining steroidogenic enzyme activities and serum T levels in rats dosed with MeHg. In conclusion, these studies show that different lipid diets can alter the toxic effects of MeHg on male rat steroidogenesis in terms of serum testosterone and steroidogenic enzyme activities.

Effects of dietary fats and proteins on rat testicular steroidogenic enzymes and serum testosterone levels.

It is known that certain dietary fats can modulate rat testosterone metabolism. In the current study we have investigated testicular steroidogenic enzyme activities and serum testosterone levels in rats fed diets containing either different protein sources (casein, fishmeal, whey) or different lipid sources (soybean oil, docosahexaenoic acid (DHA), seal oil, fish oil, lard). The diets examined reflect different marine oils and proteins which are significant components of Northern Canadian diets. Male rats (42-45 days old, 6 per group), were assigned to specific diets for 42 days. On the 43rd day of the study, rats were sacrificed and blood plasma and testes frozen (-80 degrees C) until analysis. Microsomal steroidogenic enzyme activities (3beta-HSD, 17-OHase, C-17,20-lyase, 17beta-HSD) were measured radiometrically. There were no differences in enzyme activities between the three dietary protein sources. In contrast, compared with the standard casein diet, all lipid sources caused reductions in C-17,20-lyase activity (>50%); seal oil and fish oil reduced 17-OHase activity (approximately 30%) and soybean oil, DHA fish oil and lard reduced 17beta-HSD activity (approximately 30%). No effect on 3beta-HSD activity was evident. Serum testosterone levels were determined using ELISA kits and were not affected by any diet with the exception of the soybean oil diet which was significantly elevated compared with the casein protein diet. Body and testis weights were not affected by diet. In conclusion, these data demonstrate that some dietary lipid sources caused reductions in testicular 17-OHase and C-17,20-lyase activities but not to the extent that serum T levels were affected, while soybean oil caused elevated serum testosterone in the absence of elevated steroidogenic enzyme activities.

Organotin speciation and tissue distribution in rat dams, fetuses, and neonates following oral administration of tributyltin chloride.

Tributyltin (TBT) is a biocide that contaminates human foodstuffs, especially shellfish. TBT is an endocrine disrupter, producing imposex in several marine gastropods. Previous studies showed that oral dosing of rat dams with TBT chloride leads to abnormal fetal and postnatal development. In this study, the tissue distribution and speciation of organotins in tissues were examined in dams, fetuses, and neonates following dosing of rat dams commencing on gestational day (GD) 8 by oral gavage with TBT in olive oil at 0, 0.25, 2.5, or 10 mg/kg body weight (BW)/d. Dams' body weights were significantly reduced by the 10-mg/kg BW/d TBT treatment. At GD20, there were no significant effects of any TBT treatment on pup weights, litter size, sex ratio, or tissue weights. However, at postnatal day (PND) 6 and 12, neonatal pup weights were reduced by the 10-mg/kg BW/d TBT treatment but tissue weights were unaffected, except for the liver weight of female pups, which was reduced by the 10-mg/kg BW/d TBT treatment. Tissues harvested on GD20 and PND6 and PND12 were extracted for determination of organotins by gas chromatography-atomic emission detection (GC-AED). In most tissues, TBT and its metabolite dibutyltin (DBT) were evident but monobutyltin (MBT) was rarely measured above the detection limit. The livers and brains of fetuses contained TBT and DBT at levels that were approximately 50% of the equivalent tissues in the dams. Furthermore, these tissues appeared to preferentially absorb/retain organotins, since the concentrations were greater than were found for the total loading in whole pups. The placenta also contained relatively large quantities of TBT and DBT. Postnatally, the TBT levels in pups decreased markedly, a probable consequence of the extremely low levels of organotins in rat milk. However, DBT levels in pups livers and brains were maintained, probably due to metabolism of TBT to DBT. Similarly, while dams' spleens contained significant quantities of organotins, the pups' spleens contained smaller quantities, and these decreased rapidly between PND6 and PND12. These results show that organotins cross the placenta and accumulate in fetal tissues but that during lactation, the pups would receive minimal organotins through the milk and during this period, the levels of TBT in pups' tissues decreases rapidly. Consequently, fetuses would be at greater risk of the adverse effects of TBT, but due to the lack of transfer through milk, the risk would be reduced during the lactational period.

Bioavailability of soy isoflavones in rats Part I: application of accurate methodology for studying the effects of gender and source of isoflavones.

There are limited and controversial reports about the effects of gender and source of isoflavones on their bioavailability. Moreover, several previous studies have not used appropriate methodology to determine the bioavailability of soy isoflavones, which requires comparing the area under the plasma concentration-time curve after both oral and intravenous injection (IV) administration. Therefore, the present study was conducted to determine the bioavailability of isoflavones from different sources following both oral and IV administration in male and female rats. Three sources of isoflavones; Novasoy (a commercial supplement), a mixture of synthetic aglycones (daidzein, genistein and glycitein) and a mixture of synthetic glucosides (daidzin, genistin and glycitin) were tested. Following administration, blood samples were collected at several time points (0, 10, 30 min and 1, 2, 8, 24, 48 h post oral gavage and 0, 10, 30, 45 min and 1, 2, 3, 4, 8 h post-IV dosing) and plasma isoflavones were measured by LC/MS. Bioavailability values for daidzein, genistein and glycitein were significantly (p <0.05) higher (up to sevenfold) in Novasoy and the glucoside forms of isoflavones compared with those of the aglycone forms. Moreover, significant (p <0.05) gender differences in the bioavailability of 7-hydroxyl-3-(4'-hydroxyphenyl)-chroman (a metabolite of daidzein), glycitein and daidzein were observed for Novasoy, with higher values in male rats. In summary, the source of isoflavones and the sex of rats had significant effects on isoflavone bioavailability.

Region-specific expression of androgen and growth factor pathway genes in the rat epididymis and the effects of dual 5alpha-reductase inhibition.

Dihydrotestosterone (DHT) is the primary androgen acting in the epididymis, the site of sperm maturation. Previously, we showed that the treatment of male rats with PNU157706, an inhibitor that acts on both isoforms of 5alpha-reductase to prevent DHT formation, has effects on the expression of genes implicated in processes that create the optimal luminal microenvironment required for sperm maturation, and on sperm maturation itself. However, signaling pathways involved in regulating or mediating DHT actions in the epididymis remain largely unknown. The goals of this study were to determine the expression profiles of potential signaling systems in the epididymis and assess their DHT-dependence using two different dual 5alpha-reductase inhibitors. Rats were untreated or gavaged with vehicle, 10 mg/kg per day PNU157706 or 32 mg/kg per day FK143 for 28 days and epididymal gene expression was analyzed. Gene array analysis revealed analogous effects of FK143 on overall epididymal gene expression when compared with previous PNU157706 studies. Quantitative RT-PCR analysis of the expression of the 5alpha-reductase isozymes, androgen receptor, and members of the IGF, FGF, TGF, and VEGF families revealed novel region-specific expression profiles in the epididymis that were differentially affected by 5alpha-reductase inhibition; the two inhibitors had parallel effects. Specifically, in proximal regions, 5alpha-reductase 1, androgen receptor, and TGF-beta1 expression increased after treatment, while in distal regions expression of IGF-I, IGFBP-5, IGFBP-6, and FGF-10 decreased. These results provide insight into epididymal signaling mechanisms and indicate potential candidates acting either upstream or downstream of DHT to regulate and/or mediate its actions in the epididymis.

Effects of ciprofibrate on testicular and adrenal steroidogenic enzymes in the rat.

Testicular and adrenal steroidogenic enzymes were measured radiometrically following oral dosing of rats with ciprofibrate (2-[4-(2,2-dichlorocyclopropyl) phenoxyl]-2-methylpropinoic acid), a peroxisome proliferator. Six-week-old male Fisher 344 rats were fed a diet containing ciprofibrate (0.025%, w/w) for 3, 7, 14, 28, 56, 84, 112 or 140 days leading to a daily ciprofibrate intake of approximately 15 mg/kg body weight/day. Ciprofibrate caused a marked inhibition of testicular 3beta-hydroxysteroid dehydrogenase-isomerase (3beta-HSD) activity that was significant after 3 days and subsequently decreased to 40% of control level. Ciprofibrate treatment also reduced 17beta-hydroxysteroid dehydrogenase (17beta-HSD) activity to a lesser extent but had no effect on 17-hydroxylase (17-OHase) activity. Immunoblot analyses indicated that ciprofibrate treatment did not alter enzyme protein levels and semi-quantitative RT-PCR analysis also revealed no significant changes in testicular 3beta-HSD mRNA levels. Furthermore, in addition to the enzyme-specific effect of ciprofibrate on 3beta-HSD in the testes, a tissue-specific effect was also evident, since no significant effects of ciprofibrate were seen on the activities of 3beta-HSD or 21-OHase in the adrenal glands from the same animals.

A review of the animal models used to investigate the health benefits of soy isoflavones.

This review considers the recent literature in which animal models were used to investigate the purported health benefits of soy isoflavones. The main conclusions are that our animal models demonstrate minimal effects in breast, prostate, and colon cancer prevention, and that, while some cancers may respond to isoflavones, it would appear that isoflavones do not prevent further development once cancer has become established. Regarding cardiovascular health, the lipid-lowering effects of isoflavones have been established, but their efficacy may be less than original research purported. However, it may be considered a bonus of habitual soy consumption that blood cholesterol levels would be reduced somewhat. With respect to osteoporosis and menopausal symptoms, animal models do not show any consistent benefit of isoflavones in preventing osteoporosis, and calcium fortification or the use of prescribed medications are likely much better approaches to combat bone loss. However, our animal models of osteoporosis and menopausal symptoms may not be entirely representative of the human situation. Perhaps the benefit of isoflavones in cognitive skills and in delaying Alzheimer's disease is an area where they can be of some advantage. However, this field is very recent and requires much more research in both humans and animal models before any definitive benefit can be propounded. On the other hand, isoflavones in moderation are probably not dangerous, as few studies have indicated adverse effects. However, large doses have been shown to increase apoptosis and cell degeneration, and in some cancer regimes, once the cancer has progressed beyond the hormone-dependent stage, high doses of isoflavones may be contraindicated. The prospect of mega-dosing from isoflavone supplements opens a new chapter in the risk assessment of isoflavone consumption.

An accurate and reproducible method for the quantitative analysis of isoflavones and their metabolites in rat plasma using liquid chromatography/mass spectrometry combined with photodiode array detection.

To study the safety and potential health benefits of soy isoflavones, a rapid and simple method based on liquid chromatography combined with mass spectrometry (LC/MS) and photodiode array detector (PDA) was developed for the determination of isoflavones in rat plasma. The analytes included daidzein, genistein, glycitein, equol, 4-ethyl phenol, and biochanin A over a concentration range of 1.0-4320.0 nM using 75 microL of rat plasma. Rat plasma samples were hydrolyzed by adding an enzyme mixture from Helix pomatia containing glucuronidase and sulfatase to convert the isoflavone beta-glycosides daidzin, genistin, and glycitin to their active aglycone forms. A liquid-liquid extraction method using ethyl acetate as the extraction solvent was used to extract aglycones and the internal standards (phenolphthalein beta-D glucuronide, 4-methylumbelliferyl sulfate, and apigenin) from digested plasma samples. The extract was evaporated to dryness under a nitrogen stream, reconstituted with 0.1% formic acid in water-acetonitrile (85 + 15), and injected into a Zorbax SB-CN reversed-phase column (4.6 x 75 mm, 3.5 microm particle size). The Micromass ZQ detector was operated in the positive ion selected-ion monitoring mode. The flow rate for LC was 1.0 mL/min, with a split where 25% of the effluent was introduced into the electrospray ionization probe of the MS instrument and 75% into the PDA. The chromatographic run time was 16.0 min, with delay of 10 min/injection. The interday precision and accuracy of the standard samples were <2.6% relative standard deviation and <10% relative error, respectively. Recovery of the reported isoflavones with this method varied from 86 to 100%.