Characterizing biopersistence potential of the metabolite 5:3 fluorotelomer carboxylic acid after repeated oral exposure to the 6:2 fluorotelomer alcohol.

Our previous report on pharmacokinetic (PK) evaluation of 6:2 fluorotelomer alcohol (6:2 FTOH) examined the biopersistence potential of its metabolites based on data published from single inhalation and occupational 6:2 FTOH exposure studies. We calculated internal exposure estimates of three key metabolites of 6:2 FTOH, of which 5:3 fluorotelomer carboxylic acid (5:3 acid) had the highest internal exposure and the slowest clearance. No oral repeated 6:2 FTOH exposure data were available at the time to fully characterize the biopersistence potential of the metabolite 5:3 acid. We recently received additional data on 6:2 FTOH and 5:3 acid, which included a 90-day toxicokinetic study report on repeated oral 6:2 FTOH exposure to rats. We reviewed the study and analyzed the reported 5:3 acid concentrations in plasma, liver, and fat using one-compartment PK modeling and calculated elimination rate constants (kel), elimination half-lives (t1/2) and times to steady state (tss) of 5:3 acid at three 6:2 FTOH doses. Our results showed that tss of 5:3 acid in plasma and evaluated tissues were approximately close to 1 year, such that the majority of highest values were observed at the lowest 6:2 FTOH dose, indicating its association with the biopersistence of 6:2 FTOH. The results of our PK analysis are the first to characterize biopersistence potential of the 5:3 acid after repeated oral exposure to the parent compound 6:2 FTOH based on steady state PK parameters, and therefore, may have an impact on future study designs when conducting toxicity assays for such compounds.

Licorice root components mimic estrogens in an object location task but not an object recognition task.

This study investigated the efficacy of components of licorice root to alter performance on two different recognition tasks, a hippocampus-sensitive metric change in object location (MCOL) task and a striatum-sensitive double object recognition (DOR) task. Isoliquiritigenin (ISL), licorice root extract (LRE), and whole licorice root powder (LRP) were assessed. Young adult female rats were ovariectomized (OVX) and exposed to ISL, LRE or LRP at 0.075%, 0.5% or 5% respectively in the diet. An estradiol group was included as a positive control based on our prior findings. Rats were allowed to explore two objects for three 5-min study trials (separated by 3-min intervals) before a fourth 5-min test trial where the objects were moved closer together (MCOL task) or replaced with two new objects (DOR task). Rats typically habituate to the objects across the three study trials. An increase in object exploration time in the test trial suggests the rat detected the change. Estradiol improved MCOL performance and impaired DOR performance, similar to previously shown effects of estradiol and other estrogens, which tend to improve learning and memory on hippocampus-sensitive tasks and impair striatum-sensitive cognition. LRP had no effect on recognition while exposure to ISL and LRE improved MCOL performance. Exposure to ISL, LRE and LRP failed to attenuate DOR, contrary to effects of estradiol shown here and to previous reports in young-adult OVX rats. These findings suggest components of licorice root may prove to be effective therapies targeting memory enhancement without unintended deleterious cognitive effects.

Furan-induced transcriptomic and gene-specific DNA methylation changes in the livers of Fischer 344 rats in a 2-year carcinogenicity study.

Furan is a significant food contaminant and a potent hepatotoxicant and rodent liver carcinogen. The carcinogenic effect of furan has been attributed to genotoxic and non-genotoxic, including epigenetic, changes in the liver; however, the mechanisms of the furan-induced liver tumorigenicity are still unclear. The goal of the present study was to investigate the role of transcriptomic and epigenetic events in the development of hepatic lesions in Fischer (F344) rats induced by furan treatment in a classic 2-year rodent tumorigenicity bioassay. High-throughput whole-genome transcriptomic analysis demonstrated distinct alterations in gene expression in liver lesions induced in male F344 rats treated with 0.92 or 2.0 mg furan/kg body weight (bw)/day for 104 weeks. Compared to normal liver tissue, 1336 and 1541 genes were found to be differentially expressed in liver lesions in rats treated with 0.92 and 2.0 mg furan/kg bw/day, respectively, among which 1001 transcripts were differentially expressed at both doses. Pairing transcriptomic and next-generation bisulfite sequencing analyses of the common differentially expressed genes identified 42 CpG island-containing genes in which the methylation level was correlated inversely with gene expression. Forty-eight percent of these genes (20 genes, including Areg, Jag1, and Foxe1) that exhibited the most significant methylation and gene expression changes were involved in key pathways associated with different aspects of liver pathology. Our findings illustrate that gene-specific DNA methylation changes have functional consequences and may be an important component of furan hepatotoxicity and hepatocarcinogenicity.

Development of a physiologically based pharmacokinetic model for assessment of human exposure to bisphenol A.

A previously developed physiologically based pharmacokinetic (PBPK) model for bisphenol A (BPA) in adult rhesus monkeys was modified to characterize the pharmacokinetics of BPA and its phase II conjugates in adult humans following oral ingestion. Coupled with in vitro studies on BPA metabolism in the liver and the small intestine, the PBPK model was parameterized using oral pharmacokinetic data with deuterated-BPA (d6-BPA) delivered in cookies to adult humans after overnight fasting. The availability of the serum concentration time course of unconjugated d6-BPA offered direct empirical evidence for the calibration of BPA model parameters. The recalibrated PBPK adult human model for BPA was then evaluated against published human pharmacokinetic studies with BPA. A hypothesis of decreased oral uptake was needed to account for the reduced peak levels observed in adult humans, where d6-BPA was delivered in soup and food was provided prior to BPA ingestion, suggesting the potential impact of dosing vehicles and/or fasting on BPA disposition. With the incorporation of Monte Carlo analysis, the recalibrated adult human model was used to address the inter-individual variability in the internal dose metrics of BPA for the U.S. general population. Model-predicted peak BPA serum levels were in the range of pM, with 95% of human variability falling within an order of magnitude. This recalibrated PBPK model for BPA in adult humans provides a scientific basis for assessing human exposure to BPA that can serve to minimize uncertainties incurred during extrapolations across doses and species.

24-hour human urine and serum profiles of bisphenol A: Evidence against sublingual absorption following ingestion in soup.

Extensive first-pass metabolism of ingested bisphenol A (BPA) in the gastro-intestinal tract and liver restricts blood concentrations of bioactive BPA to <1% of total BPA in humans and non-human primates. Absorption of ingested BPA through non-metabolizing tissues of the oral cavity, recently demonstrated in dogs, could lead to the higher serum BPA concentrations reported in some human biomonitoring studies. We hypothesized that the extensive interaction with the oral mucosa by a liquid matrix, like soup, relative to solid food or capsules, might enhance absorption through non-metabolizing oral cavity tissues in humans, producing higher bioavailability and higher serum BPA concentrations. Concurrent serum and urine concentrations of d6-BPA, and its glucuronide and sulfate conjugates, were measured over a 24hour period in 10 adult male volunteers following ingestion of 30µg d6-BPA/kg body weight in soup. Absorption of d6-BPA was rapid (t1/2=0.45h) and elimination of the administered dose was complete 24h post-ingestion, evidence against any tissue depot for BPA. The maximum serum d6-BPA concentration was 0.43nM at 1.6h after administration and represented <0.3% of total d6-BPA. Pharmacokinetic parameters, pharmacokinetic model simulations, and the significantly faster appearance half-life of d6-BPA-glucuronide compared to d6-BPA (0.29h vs 0.45h) were evidence against meaningful absorption of BPA in humans through any non-metabolizing tissue (<1%). This study confirms that typical exposure to BPA in food produces picomolar to subpicomolar serum BPA concentrations in humans, not nM concentrations reported in some biomonitoring studies.

Testicular development in male rats is sensitive to a soy-based diet in the neonatal period.

Approximately 30% of infants in the United States are exposed to high doses of isoflavones resulting from soy infant formula consumption. Soybeans contain the isoflavones genistin and daidzin, which are hydrolyzed in the gastrointestinal tract to their genistein and daidzein aglycones. Both aglycones possess hormonal activity and may interfere with male reproductive development. Testosterone, which supports male fertility, is mainly produced by testicular Leydig cells. Our previous studies indicated that perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells and increased testosterone concentrations into adulthood. However, the relevance of the neonatal period as part of the perinatal window of isoflavone exposure remains to be established. The present study examined the effects of exposure to isoflavones on male offspring of dams maintained on a casein-based control or whole soybean diet in the neonatal period, that is, Days 2 to 21 postpartum. The results showed that the soybean diet stimulated proliferative activity in developing Leydig cells while suppressing their steroidogenic capacity in adulthood. In addition, isoflavone exposure decreased production of anti-Müllerian hormone by Sertoli cells. Similar to our previous in vitro studies of genistein action in Leydig cells, daidzein induced proliferation and interfered with signaling pathways to suppress steroidogenic activity. Overall, the data showed that the neonatal period is a sensitive window of exposure to isoflavones and support the view that both genistein and daidzein are responsible for biological effects associated with soy-based diets.

Reaction of dehydropyrrolizidine alkaloids with valine and hemoglobin.

Pyrrolizidine alkaloid-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids exert toxicity through metabolism to dehydropyrrolizidine alkaloids that bind to cellular protein and DNA, leading to hepatotoxicity, genotoxicity, and tumorigenicity. To date, it is not clear how dehydropyrrolizidine alkaloids bind to cellular constituents, including amino acids and proteins, resulting in toxicity. Metabolism of carcinogenic monocrotaline, riddelliine, and heliotrine produces dehydromonocrotaline, dehyroriddelliine, and dehydroheliotrine, respectively, as primary reactive metabolites. In this study, we report that reaction of dehydromonocrotaline with valine generated four highly unstable 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived valine (DHP-valine) adducts. For structural elucidation, DHP-valine adducts were derivatized with phenyl isothiocyanate (PITC) to DHP-valine-PITC products. After HPLC separation, their structures were characterized by mass spectrometry, UV-visible spectrophotometry, (1)H NMR, and (1)H-(1)H COSY NMR spectral analysis. Two DHP-valine-PITC adducts, designated as DHP-valine-PITC-1 and DHP-valine-PITC-3, had the amino group of valine linked to the C7 position of the necine base, and the other two DHP-valine-PITC products, DHP-valine-PITC-2 and DHP-valine-PITC-4, linked to the C9 position of the necine base. DHP-valine-PITC-1 was interconvertible with DHP-valine-PITC-3, and DHP-valine-PITC-2 was interconvertible with DHP-valine-PITC-4. Reaction of dehydroriddelliine and dehydroheliotrine with valine provided similar results. However, reaction of valine and dehydroretronecine (DHR) under similar experimental conditions did not produce DHP-valine adducts. Reaction of dehydromonocrotaline with rat hemoglobin followed by derivatization with PITC also generated the same four DHP-valine-PITC adducts. This represents the first full structural elucidation of protein conjugated pyrrolic adducts formed from reaction of dehydropyrrolizidine alkaloids with an amino acid (valine). In addition, it was found that DHP-valine-2 and DHP-valine-4, with the valine amino group linked at the C7 position of the necine base, can lose the valine moiety to form DHP.

Evaluating the toxicokinetics of some metabolites of a C6 polyfluorinated compound, 6:2 fluorotelomer alcohol in pregnant and nonpregnant rats after oral exposure to the parent compound.

The 6:2 fluorotelomer alcohol (6:2 FTOH) is a common impurity in per- and polyfluoroalkyl substances (PFASs) used in many applications. Our previous toxicokinetic (TK) evaluation of 6:2 FTOH calculated times to steady state (tss) of one of its metabolites, 5:3 fluorotelomer carboxylic acid (5:3A), in the plasma and tissues of up to a year after oral exposure to rats. Our current work further elucidated the TK of 5:3A and other metabolites of 6:2 FTOH in pregnant and nonpregnant rats after repeated oral exposure and examined the role of renal transporters in the biopersistence of 5:3A. The tss values for 5:3A in serum and tissues of adult nonpregnant animals ranged from 150 days to over a year. 4:3 fluorotelomer carboxylic acid (4:3A) was an additional potentially-biopersistent metabolite. 5:3A was the major metabolite of 6:2 FTOH in serum of pregnant dams and fetuses at each time interval. 5:3A was not a substrate for renal transporters in a human kidney cell line in vitro, indicating that renal reuptake of 5:3A is unlikely contribute to its biopersistence. Further research is needed to identify the underlying processes and evaluate the impact of these 6:2 FTOH metabolites on human health.

Prediction and evaluation of route dependent dosimetry of BPA in rats at different life stages using a physiologically based pharmacokinetic model.

Bisphenol A (BPA) has received considerable attention throughout the last decade due to its widespread use in consumer products. For the first time a physiologically based pharmacokinetic (PBPK) model was developed in neonatal and adult rats to quantitatively evaluate age-dependent pharmacokinetics of BPA and its phase II metabolites. The PBPK model was calibrated in adult rats using studies on BPA metabolism and excretion in the liver and gastrointestinal tract, and pharmacokinetic data with BPA in adult rats. For immature rats the hepatic and gastrointestinal metabolism of BPA was inferred from studies on the maturation of phase II enzymes coupled with serum time course data in pups. The calibrated model predicted the measured serum concentrations of BPA and BPA conjugates after administration of 100µg/kg of d6-BPA in adult rats (oral gavage and intravenous administration) and postnatal days 3, 10, and 21 pups (oral gavage). The observed age-dependent BPA serum concentrations were partially attributed to the immature metabolic capacity of pups. A comparison of the dosimetry of BPA across immature rats and monkeys suggests that dose adjustments would be necessary to extrapolate toxicity studies from neonatal rats to infant humans.

Concurrent determination of bisphenol A pharmacokinetics in maternal and fetal rhesus monkeys.

Bisphenol A (BPA) is an important industrial chemical used as the monomer for polycarbonate plastic and in epoxy resins for food can liners. Worldwide biomonitoring studies consistently find a high prevalence of BPA conjugates in urine (>90%) in amounts consistent with aggregate exposure at levels below 1 µg/kg bw/d. The current study used LC/MS/MS to measure concurrently the pharmacokinetics of aglycone (active) and conjugated (inactive) deuterated BPA (d6) in maternal and fetal rhesus monkey serum, amniotic fluid, and placenta following intravenous injection in the dam (100 µg/kg bw). Internal exposures of the fetus to aglycone d6-BPA (serum AUC) were attenuated by maternal, placental, and fetal Phase II metabolism to less than half that in the dam. Levels of aglycone and conjugated d6-BPA measured in whole placenta were consistent with a role in metabolic detoxification. The monotonic elimination of aglycone d6-BPA from the fetal compartment accompanied by persistent conjugate levels provides further evidence arguing against the hypothesis that BPA conjugates are selectively deconjugated by either the placenta or fetus. These results also provide benchmarks to guide the interpretation of human cord blood, amniotic fluid, and placenta sampling and measurement strategies as a basis for estimating fetal exposures to BPA. This study in a non-human primate model provides additional pharmacokinetic data for use in PBPK modeling of perinatal exposures to BPA from food contact, medical devices, and other environmental sources.

Recent advances in the risk assessment of melamine and cyanuric acid in animal feed.

Melamine can be present at low levels in food and feed mostly from its legal use as a food contact material in laminates and plastics, as a trace contaminant in nitrogen supplements used in animal feeds, and as a metabolite of the pesticide cyromazine. The mechanism of toxicity of melamine involves dose-dependent formation of crystals with either endogenous uric acid or a structural analogue of melamine, cyanuric acid, in renal tubules resulting in potential acute kidney failure. Co-exposure to melamine and cyanuric acid in livestock, fish, pets and laboratory animals shows higher toxicity compared with melamine or cyanuric acid alone. Evidence for crystal formation between melamine and other structural analogs i.e. ammelide and ammeline is limited. Illegal pet food adulterations with melamine and cyanuric acid and adulteration of milk with melamine resulted in melamine-cyanuric acid crystals, kidney damage and deaths of cats and dogs and melamine-uric acid stones, hospitalisation and deaths of children in China respectively. Following these incidents, the tolerable daily intake for melamine was re-evaluated by the U.S. Food and Drug Administration, the World Health Organisation, and the Scientific Panel on Contaminants in the Food Chain of the European Food Safety Authority (EFSA). This review provides an overview of toxicology, the adulteration incidents and risk assessments for melamine and its structural analogues. Particular focus is given to the recent EFSA risk assessment addressing impacts on animal and human health of background levels of melamine and structural analogues in animal feed. Recent research and future directions are discussed.

Pyrrolizidine alkaloid-derived DNA adducts as a common biological biomarker of pyrrolizidine alkaloid-induced tumorigenicity.

Pyrrolizidine alkaloid-containing plants are the most common poisonous plants affecting livestock, wildlife, and humans. The U.S. National Toxicology Program (NTP) classified riddelliine, a tumorigenic pyrrolizidine alkaloid, as "reasonably anticipated to be a human carcinogen" in the NTP 12th Report on Carcinogens in 2011. We previously determined that four DNA adducts were formed in rats dosed with riddelliine. The structures of the four DNA adducts were elucidated as (i) a pair of epimers of 7-hydroxy-9-(deoxyguanosin-N(2)-yl)dehydrosupinidine adducts (termed as DHP-dG-3 and DHP-dG-4) as the predominant adducts; and (ii) a pair of epimers of 7-hydroxy-9-(deoxyadenosin-N(6)-yl)dehydrosupinidine adducts (termed as DHP-dA-3 and DHP-dA-4 adducts). In this study, we selected a nontumorigenic pyrrolizidine alkaloid, platyphylliine, a pyrrolizidine alkaloid N-oxide, riddelliine N-oxide, and nine tumorigenic pyrrolizidine alkaloids (riddelliine, retrorsine, monocrotaline, lycopsamine, retronecine, lasiocarpine, heliotrine, clivorine, and senkirkine) for study in animals. Seven of the nine tumorigenic pyrrolizidine alkaloids, with the exception of lycopsamine and retronecine, are liver carcinogens. At 8-10 weeks of age, female F344 rats were orally gavaged for 3 consecutive days with 4.5 and 24 µmol/kg body weight test article in 0.5 mL of 10% DMSO in water. Twenty-four hours after the last dose, the rats were sacrificed, livers were removed, and liver DNA was isolated for DNA adduct analysis. DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4 adducts were formed in the liver of rats treated with the individual seven hepatocarcinogenic pyrrolizidine alkaloids and riddelliine N-oxide. These DNA adducts were not formed in the liver of rats administered retronecine, the nontumorigenic pyrrolizidine alkaloid, platyphylliine, or vehicle control. These results indicate that this set of DNA adducts, DHP-dG-3, DHP-dG-4, DHP-dA-3, and DHP-dA-4, is a common biological biomarker of pyrrolizidine alkaloid-induced liver tumor formation. To date, this is the first finding that a set of exogenous DNA adducts are commonly formed from a series of tumorigenic xenobiotics.

Low-dose dietary genistein negates the therapeutic effect of tamoxifen in athymic nude mice.

The present study examined the effect of dietary genistein, a soy isoflavone, on breast cancer patients who take tamoxifen, an antiestrogen treatment, using a preclinical model. The interaction of various doses of genistein with tamoxifen on the growth of estrogen receptor-positive breast cancer MCF-7 cells was investigated by subcutaneously injecting MCF-7 cells into the flank of ovariectomized athymic mice. Animals were randomized into eight experimental groups with 10-13 mice per group: control (C), estrogen (E) (0.08 mg E implant), tamoxifen (T) (3 mg T implant), estrogen + tamoxifen (E + T), tamoxifen + 500 p.p.m. genistein (T + G500), estrogen + tamoxifen + 250 p.p.m. genistein (E + T + G250), estrogen + tamoxifen + 500 p.p.m. genistein (E + T + G500) and estrogen + tamoxifen + 1000 p.p.m. genistein (E + T + G1000). Treatment of tamoxifen significantly reduced the estrogen-induced MCF-7 tumor prevalence and tumor size. This inhibitory effect of tamoxifen was significantly negated by the low doses of dietary genistein (250 and 500 p.p.m.), whereas the 1000 p.p.m. genistein did not have the same effect. Cells harvested from tamoxifen-treated tumors retained estrogen responsiveness of their progenitor MCF-7 cells, indicating that the abrogating effect of genistein on tamoxifen-treated tumor growth was not caused by a diminished tamoxifen response but directly by genistein. The low doses of dietary genistein abrogated the inhibitory effect of tamoxifen potentially by acting on the tumor cell proliferation/apoptosis ratio and the messenger RNA (mRNA) expression of cyclin D1 in addition to regulating the mRNA expression of progesterone receptor. Therefore, data from the current study suggest that caution is warranted regarding the consumption of dietary genistein by breast cancer patients while on tamoxifen therapy.

Tumorigenicity of acrylamide and its metabolite glycidamide in the neonatal mouse bioassay.

Acrylamide is a high-volume industrial chemical, a component of cigarette smoke, and a product formed in certain foods prepared at high temperatures. Previously, we compared the extent of DNA adduct formation and mutations in B6C3F(1) /Tk mice treated neonatally with acrylamide or glycidamide to obtain information concerning the mechanism of acrylamide genotoxicity. We have now examined the tumorigenicity of acrylamide and glycidamide in mice treated neonatally. Male B6C3F(1) mice were injected intraperitoneally on postnatal days 1, 8 and 15 with 0.0, 0.14 or 0.70 mmol acrylamide or glycidamide per kg body weight per day and the tumorigenicity was assessed after 1 year. Survival in each of the groups was >87%, there were no differences in body weights among the groups, and the only treatment-related neoplasms involved the liver. The incidence of combined hepatocellular adenoma or carcinoma was 3.8% in the control group, 8.3% in the 0.14 mmol acrylamide and glycidamide per kg body weight groups, 4.2% in the 0.70 mmol acrylamide per kg body weight group and 71.4% in the 0.70 mmol glycidamide per kg body weight group. Analysis of the hepatocellular tumors indicated that the increased incidence observed in mice administered 0.70 mmol glycidamide per kg body weight was associated with A → G and A → T mutations at codon 61 of H-ras. These results, combined with our previous data on DNA adduct formation and mutation induction, suggest that the carcinogenicity of acrylamide is dependent on its metabolism to glycidamide, a pathway that is deficient in neonatal mice.

In vivo genotoxicity of furan in F344 rats at cancer bioassay doses.

Furan, a potent rodent liver carcinogen, is found in many cooked food items and thus represents a human cancer risk. Mechanisms for furan carcinogenicity were investigated in male F344 rats using the in vivo Comet and micronucleus assays, combined with analysis of histopathological and gene expression changes. In addition, formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III (EndoIII)-sensitive DNA damage was monitored as a measure of oxidative DNA damage. Rats were treated by gavage on four consecutive days with 2, 4, and 8mg/kg bw furan, doses that were tumorigenic in 2-year cancer bioassays, and with two higher doses, 12 and 16mg/kg. Rats were killed 3h after the last dose, a time established as producing maximum levels of DNA damage in livers of furan-treated rats. Liver Comet assays indicated that both DNA strand breaks and oxidized purines and pyrimidines increased in a near-linear dose-responsive fashion, with statistically significant increases detected at cancer bioassay doses. No DNA damage was detected in bone marrow, a non-target tissue for cancer, and peripheral blood micronucleus assays were negative. Histopathological evaluation of liver from furan-exposed animals produced evidence of inflammation, single-cell necrosis, apoptosis, and cell proliferation. In addition, genes related to apoptosis, cell-cycle checkpoints, and DNA-repair were expressed at a slightly lower level in the furan-treated livers. Although a mixed mode of action involving direct DNA binding cannot be ruled out, the data suggest that furan induces cancer in rat livers mainly through a secondary genotoxic mechanism involving oxidative stress, accompanied by inflammation, cell proliferation, and toxicity.

Acute genistein treatment mimics the effects of estradiol by enhancing place learning and impairing response learning in young adult female rats.

Endogenous estrogens have bidirectional effects on learning and memory, enhancing or impairing cognition depending on many variables, including the task and the memory systems that are engaged. Moderate increases in estradiol enhance hippocampus-sensitive place learning, yet impair response learning that taps dorsal striatal function. This memory modulation likely occurs via activation of estrogen receptors, resulting in altered neural function. Supplements containing estrogenic compounds from plants are widely consumed despite limited information about their effects on brain function, including learning and memory. Phytoestrogens can enter the brain and signal through estrogen receptors to affect cognition. Enhancements in spatial memory and impairments in executive function have been found following treatment with soy phytoestrogens, but no tests of actions on striatum-sensitive tasks have been made to date. The present study compared the effects of acute exposure to the isoflavone genistein with the effects of estradiol on performance in place and response learning tasks. Long-Evans rats were ovariectomized, treated with 17ß-estradiol benzoate, genistein-containing sucrose pellets, or vehicle (oil or plain sucrose pellets) for 2 days prior to behavioral training. Compared to vehicle controls, estradiol treatment enhanced place learning at a low (4.5 µg/kg) but not high dose (45 µg/kg), indicating an inverted pattern of spatial memory facilitation. Treatment with 4.4 mg of genistein over 2 days also significantly enhanced place learning over vehicle controls. For the response task, treatment with estradiol impaired learning at both low and high doses; likewise, genistein treatment impaired response learning compared to rats receiving vehicle. Overall, genistein was found to mimic estradiol-induced shifts in place and response learning, facilitating hippocampus-sensitive learning and slowing striatum-sensitive learning. These results suggest signaling through estrogen receptor ß and membrane-associated estrogen receptors in learning enhancements and impairments given the preferential binding of genistein to the ERß subtype and affinity for GPER.

Genotoxicity of furan in Big Blue rats.

Furan is a multispecies liver carcinogen whose cancer mode of action (MOA) is unclear. A major metabolite of furan is a direct acting mutagen; however, it is not known if genotoxicity is a key step in the tumors that result from exposure to furan. In order to address this question, transgenic Big Blue rats were treated by gavage five times a week for 8 weeks with two concentrations of furan used in cancer bioassays (2 and 8mg/kg), and with two higher concentrations (16 and 30mg/kg). Peripheral blood samples taken 24h after the 5th dose (1 week of dosing) were used to assay for micronucleus (MN) frequency in normochromatic erythrocytes (NCEs) and reticulocytes (RETs), and Pig-a gene mutation in total red blood cells (RBCs). 24h after the last dose of the 8-week treatment schedule, the rats were euthanized, and their tissues were used to perform NCE and RET MN assays, the Pig-a RBC assay, Pig-a and Hprt lymphocyte gene mutation assays, the liver cII transgene mutation assay, and the liver Comet assay. The responses in the MN assays conducted at both sampling times, and all the gene mutation assays, were uniformly negative; however, the Comet assay was positive for the induction of liver DNA damage. As the positive responses in the Comet assay were seen only with doses in excess of the cancer bioassay doses, and at least one of these doses (30mg/kg) produced toxicity in the liver, the overall findings from the study are consistent with furan having a predominantly nongenotoxic MOA for cancer.

Isoliquiritigenin Decreases Bone Resorption and Osteoclast Differentiation.

SCOPE: A dose-ranging study is performed using young estrogen-depleted rats to determine whether dietary isoliquiritigenin (ILQ) alters bone metabolism and if the effects are associated with estrogen receptor signaling. METHODS AND RESULTS: Six-week-old rats (ovariectomized at 4 weeks of age) are fed diets containing 0, 100, 250, or 750 ppm ILQ (n = 5/treatment) for 7 days. Gene expression in femur and uterus, blood markers of bone turnover, body composition, and uterine weight and epithelial cell height are determined. Because ILQ lowers bone resorption, the effect of ILQ on in vitro differentiation of osteoclasts from bone marrow of mice is assessed. Treatment resulted in a dose-dependent increases in serum ILQ but no changes in serum osteocalcin, a marker of global bone formation. Contrastingly, ILQ administration results in reduced serum CTX-1, a marker of global bone resorption, and reduces tartrate resistant acid phosphatase expression in osteoclast culture. ILQ treatment and endogenous estrogen production had limited overlap on gene expression in femur and uterus. However, uterine epithelial cell hyperplasia is observed in two of five animals treated with 750 ppm. CONCLUSIONS: In conclusion, dietary ILQ reduces bone resorption in vivo and osteoclast differentiation in vitro, by mechanisms likely differing from actions of ovarian hormones.

Pharmacokinetics and derivation of an anticancer dosing regimen for PAC-1, a preferential small molecule activator of procaspase-3, in healthy dogs.

PAC-1 is a preferential small molecule activator of procaspase-3 and has potential to become a novel and effective anticancer agent. The rational development of PAC-1 for translational oncologic applications would be advanced by coupling relevant in vitro cytotoxicity studies with pharmacokinetic investigations conducted in large mammalian models possessing similar metabolism and physiology as people. In the present study, we investigated whether concentrations and exposure durations of PAC-1 that induce cytotoxicity in lymphoma cell lines in vitro can be achievable in healthy dogs through a constant rate infusion (CRI) intravenous delivery strategy. Time- and dose-dependent procaspase-3 activation by PAC-1 with subsequent cytotoxicity was determined in a panel of B-cell lymphoma cells in vitro. The pharmacokinetics of PAC-1 administered orally or intravenously was studied in 6 healthy dogs using a crossover design. The feasibility of maintaining steady state plasma concentration of PAC-1 for 24 or 48 h that paralleled in vitro cytotoxic concentrations was investigated in 4 healthy dogs. In vitro, PAC-1 induced apoptosis in lymphoma cell lines in a time- and dose-dependent manner. The oral bioavailability of PAC-1 was relatively low and highly variable (17.8 ± 9.5%). The achievement and maintenance of predicted PAC-1 cytotoxic concentrations in normal dogs was safely attained via intravenous CRI lasting for 24 or 48 h in duration. Using the dog as a large mammalian model, PAC-1 can be safely administered as an intravenous CRI while achieving predicted in vitro cytotoxic concentrations.

Bioavailability of soy isoflavones through placental/lactational transfer and soy food.

Isoflavones are non-nutritive components of soy responsible for estrogenic responses observed in vitro and in experimental animals. Possible beneficial effects (e.g., reduction of serum lipids, increased bone mineral density, relief of hot flashes and other menopausal symptoms, mammary and prostate cancer chemoprevention) in humans have been attributed to consumption of isoflavones but evidence for potential adverse effects (e.g., stimulation of estrogen-dependent mammary tumors and aberrant perinatal development) has also been reported in experimental animal models. Bioavailability from appropriate food matrices and exposure during different life stages are both critical determinants of isoflavone effects. For these reasons, it is important to compare isoflavone bioavailability in adults to that in fetal and neonatal animals for a more complete understanding of potential susceptibility issues. Studies of the major soy isoflavone genistein were conducted in pregnant and lactating Sprague-Dawley rats to quantify placental and lactational transfer to plasma and brain to understand better biological effects observed in multigenerational studies. In addition, studies were conducted with genistein in adult Balb/c mice to define absolute bioavailability from both gavage and soy protein isolate (SPI)-containing food. The information derived from these studies makes it possible to predict internal exposures of children to genistein from soy infant formula, which is manufactured using SPI.