Michelle Vanlandingham

Pharmacokinetics of bisphenol a in neonatal and adult Sprague-Dawley rats.

Bisphenol A (BPA) is an important industrial chemical used in the manufacture of polycarbonate plastic products and epoxy resin-based food can liners. The presence of BPA in urine of >90% of Americans aged 6-60 suggests ubiquitous and frequent exposure. The current study used LC/MS/MS to measure serum pharmacokinetics of aglycone (active) and conjugated (inactive) BPA in adult and neonatal Sprague-Dawley rats by oral and injection routes. Deuterated BPA was used to avoid issues of background contamination. Linear pharmacokinetics were observed in adult rats treated orally in the range of 0-200 microg/kg bw. Evidence for enterohepatic recirculation of conjugated, but not aglycone, BPA was observed in adult rats. Significant inverse relationships were observed between postnatal age and measures of internal exposures to aglycone BPA and its elimination. In neonatal rats treated orally, internal exposures to aglycone BPA were substantially lower than from subcutaneous injection. The results reinforce the critical role for first-pass Phase II metabolism of BPA in gut and liver after oral exposure that attenuates internal exposure to the aglycone form in rats of all ages. The internal exposures to aglycone BPA observed in adult and neonatal rats following a single oral dose of 100 microg/kg bw are inconsistent with effects mediated by classical estrogen receptors based on binding affinities. However, an impact on alternative estrogen signaling pathways that have higher receptor affinity cannot be excluded in neonatal rats. These findings emphasize the importance of matching aglycone BPA internal dosimetry with receptor affinities in experimental animal studies reporting toxicity.

Distribution of bisphenol A into tissues of adult, neonatal, and fetal Sprague-Dawley rats.

Bisphenol A (BPA) is an important industrial chemical used in the manufacture of polycarbonate plastic products and epoxy resin-based food can liners. The presence of BPA metabolites in urine of >90% of Americans aged 6-60 suggests ubiquitous and frequent exposure in the range of 0.02-0.2μg/kgbw/d (25th-95th percentiles). The current study used LC/MS/MS to measure placental transfer and concentrations of aglycone (receptor-active) and conjugated (inactive) BPA in tissues from Sprague-Dawley rats administered deuterated BPA (100μg/kg bw) by oral and IV routes. In adult female rat tissues, the tissue/serum concentration ratios for aglycone BPA ranged from 0.7 in liver to 5 in adipose tissue, reflecting differences in tissue perfusion, composition, and metabolic capacity. Following IV administration to dams, placental transfer was observed for aglycone BPA into fetuses at several gestational days (GD), with fetal/maternal serum ratios of 2.7 at GD 12, 1.2 at GD 16, and 0.4 at GD 20; the corresponding ratios for conjugated BPA were 0.43, 0.65, and 3.7. These ratios were within the ranges observed in adult tissues and were not indicative of preferential accumulation of aglycone BPA or hydrolysis of conjugates in fetal tissue in vivo. Concentrations of aglycone BPA in GD 20 fetal brain were higher than in liver or serum. Oral administration of the same dose did not produce measurable levels of aglycone BPA in fetal tissues. Amniotic fluid consistently contained levels of BPA at or below those in maternal serum. Concentrations of aglycone BPA in tissues of neonatal rats decreased with age in a manner consistent with the corresponding circulating levels. Phase II metabolism of BPA increased with fetal age such that near-term fetus was similar to early post-natal rats. These results show that concentrations of aglycone BPA in fetal tissues are similar to those in other maternal and neonatal tissues and that maternal Phase II metabolism, especially following oral administration, and fetal age are critical in reducing exposures to the fetus.

Pharmacokinetics of Bisphenol A in neonatal and adult CD-1 mice: Inter-species comparisons with Sprague-Dawley rats and rhesus monkeys

Bisphenol A (BPA) is an important industrial chemical used in the manufacture of polycarbonate plastic products and epoxy resin-based food can liners. The presence of BPA metabolites in urine of >90% of Americans aged 6-60 suggests ubiquitous and frequent exposure at levels largely below 1 μg/kg bw/d. The current study used LC/MS/MS to measure serum pharmacokinetics of unconjugated (active) and conjugated (inactive) BPA in adult and neonatal CD-1 mice by oral and subcutaneous (SC) injection routes. Deuterated BPA was used to avoid issues of background contamination. Significant inverse relationships were observed between postnatal age and measures of internal exposures (C(max)) to unconjugated BPA after oral administration. Phase II conjugation, area under the time-concentration curve (AUC), and elimination half-time of unconjugated BPA were also inversely related to age. In postnatal day (PND) 3 mice, the combination of under-developed Phase II metabolism, rapid absorption, and slow elimination kinetics led to equivalent internal exposures for unconjugated BPA from oral and SC routes; however, maturing metabolic capabilities in PND 10 and older mice, led to large and significant route effects. The significant inverse age-related developmental profiles from PND 3 through adulthood for unconjugated BPA internal exposure metrics from oral administration to CD-1 mice and Sprague-Dawley rats were remarkably similar; however, the developmental profile was quite different for neonatal rhesus monkeys in which small insignificant age-related differences were observed. These results suggest that an adverse effect from BPA observed in rodent models, attributable to exposure during a discrete time period of neonatal development, would be less likely for comparable neonatal primate dosing based on internal dosimetry. On the other hand in all adults of all species studied, including humans, a low oral dose of BPA produced similarly small internal exposures for the unconjugated form, reflecting the dominant effect of presystemic Phase II metabolism.

Lactational transfer of bisphenol A in Sprague-Dawley rats.

Bisphenol A (BPA), an important industrial chemical to which humans are exposed on a daily basis, has long been associated with endocrine disruption in experimental animal models. Such exposures are of concern, particularly during fetal and early neonatal periods, because of greater vulnerability of developing organs to aberrant endocrine signaling. Although rarely reported, information about internal exposures to the receptor-active aglycone form of BPA during the perinatal period is essential to accurate assessment of potential risks. Lactating Sprague-Dawley dams were treated by daily gavage with 100 μg/kg bw d6-BPA starting at birth. Conjugated and aglycone forms of BPA were then measured by using LC/MS/MS in milk from lactating dams on PND 7 and in serum from dams and their pups on PND 10. All samples were collected 1h after dosing, a time selected to produce nearly maximal levels. While aglycone BPA was detected in all dam serum and milk samples, none was detected in pup serum (<0.2 nM). Doses delivered to pups lactationally, estimated from milk concentrations and body weights, were 300-fold lower than the dose administered to the dams. Similarly, serum concentrations of total BPA in pups were 300-fold lower than those in their dams. Furthermore, plasma concentrations of total BPA in PND 10 rat pups were 500-fold lower than peak levels achieved following direct oral delivery of the same dose to the same age pups. These findings of significant dose attenuation for the active aglycone form of BPA, relative to that of the dam, suggest high potency for toxicological effects derived exclusively from lactational transfer. Alternatively, studies that include lactational exposure and report minimal effects from BPA should consider the possibility that inadequate internal exposures were achieved during the critical postnatal period.

Comparison of Life-Stage-Dependent Internal Dosimetry for Bisphenol A, Ethinyl Estradiol, a Reference Estrogen, and Endogenous Estradiol to Test an Estrogenic Mode of Action in Sprague Dawley Rats

Bisphenol A (BPA) was administered by gavage (2.5-300,000 μg/kg body weight (bw)/day) to pregnant Sprague Dawley dams, newborn pups, and continuing into adulthood. Aglycone (i.e., unconjugated and active) and conjugated (i.e., inactive) BPA were evaluated by liquid chromatography electrospray tandem mass spectrometry (LC-ES/MS/MS) in serum to better interpret toxicological endpoints measured in the study. Ethinyl estradiol (EE2, 0.5 and 5 μg/kg bw/day) and the endogenous hormones, 17β-estradiol (E2) and testosterone, were similarly evaluated. Mean BPA aglycone levels in vehicle and naïve control rat serum (0.02-0.5 ng/ml) indicated sample processing artifact, consistent with literature reports of a propensity for postexposure blood contamination by BPA. Direct measurements of BPA-glucuronide in vehicle and naïve control serum (2-10nM) indicated unintentional exposure and metabolism at levels similar to those produced by 2.5 μg/kg bw/day BPA (7-10nM), despite careful attention to potential BPA inputs (diet, drinking water, vehicle, cages, bedding, and dust) and rigorous dosing solution certification and delivery. The source of this exposure could not be identified, but interpretation of the toxicological effects, observed only at the highest BPA doses, was not compromised. Internal exposures to BPA and EE2 aglycones were highest in young rats. When maximal serum concentrations from the two highest BPA doses and both EE2 doses were compared with concurrent levels of endogenous E2, the ERα binding equivalents were similar to or above those of endogenous E2 in male and female rats of all ages tested. Such evaluations of estrogenic internal dosimetry and comprehensive evaluation of contamination impact should aid in extrapolating risks from human BPA exposures.

Quantification of deuterated bisphenol A in serum, tissues, and excreta from adult Sprague‐Dawley rats using liquid chromatography with tandem mass spectrometry

Bisphenol A (BPA) is an important industrial chemical used in the manufacture of polycarbonate plastic products, epoxy resin-based food can liners, and paper products. The presence of BPA in urine of >90% of Americans aged 6-60 suggests ubiquitous and frequent exposure and is problematic because of the potential for endocrine disruption. The ubiquity of environmental BPA in common laboratory supplies used for sample collection, storage, and analysis greatly increases the likelihood of false positive determinations, particularly at trace levels. The current study validated using liquid chromatography/tandem mass spectrometry (LC/MS/MS) in conjunction with deuterated BPA as the dosing material to circumvent contamination for high sensitivity quantifications in rat serum, tissues, urine, and feces. The methods described provided measurements of both estrogen receptor-active aglycone and metabolically deactivated conjugated forms of BPA, a distinction that is critical to assessing toxicological potential. The adequacy of the described methodology was substantiated by its utility in analyzing samples from rats treated orally with a 100u2009µg/kg body weight dose of d6-BPA. These results emphasize the challenges inherent in measuring BPA in biological samples and how employing stable isotope labeled dosing can facilitate pharmacokinetic studies needed to understand BPA metabolism and disposition. Such studies conducted in experimental animal models, in conjunction with properly validated human biomonitoring data, will be the basis for PBPK modeling of BPA in environmentally exposed humans.

Pharmacokinetics of bisphenol A in serum and adipose tissue following intravenous administration to adult female CD-1 mice

Bisphenol A (BPA) is an important industrial chemical used as the monomer for polycarbonate plastic and in epoxy resins for use in food can liners. Worldwide biomonitoring studies consistently find high prevalence of BPA conjugates in urine consistent with pervasive exposure at levels typically below 1 μg/kg bw/day. The current study used LC/MS/MS to measure serum pharmacokinetics of unconjugated (active) and conjugated (inactive) BPA in adult female CD-1 mice following intravenous (IV) injection, which produces higher serum levels by circumventing the processes of absorption from the GI tract and presystemic metabolism that occur after oral administration. Deuterated BPA (100 μg/kg bw) was used to avoid interference by background contamination from trace amounts of native BPA. Additionally, the pharmacokinetics of unconjugated BPA were determined in adipose tissue, a proposed site of action and depot for BPA. After IV injection, unconjugated BPA rapidly distributed out of the circulation (t(1/2)=0.2 h) and terminal elimination also proceeded rapidly (t(1/2)=0.8 h). Consistent with the degree of aqueous solubility, lipid/water solubility ratio, and partitioning from blood into adipose tissue in vivo, the levels of unconjugated BPA in mouse adipose tissue rapidly reached a maximal level (0.25 h) that did not exceed the serum maximum at the initial sampling time (0.08 h). Terminal elimination of unconjugated BPA from adipose tissue (t(1/2)=7.0 h) was similar to that for conjugated BPA in serum (t(1/2)=6.6 h) and <0.01% of the administered dose remained in adipose tissue after 24 h. These plasma and tissue kinetics are consistent with rapid equilibria and underscore the non-persistent nature of BPA, particularly when compared with slowly metabolized lipophilic organic pollutants like halogenated dibenzodioxins.

NIEHS/FDA CLARITY-BPA research program update.

Bisphenol A (BPA) is a chemical used in the production of numerous consumer products resulting in potential daily human exposure to this chemical. The FDA previously evaluated the body of BPA toxicology data and determined that BPA is safe at current exposure levels. Although consistent with the assessment of some other regulatory agencies around the world, this determination of BPA safety continues to be debated in scientific and popular publications, resulting in conflicting messages to the public. Thus, the National Toxicology Program (NTP), National Institute of Environmental Health Sciences (NIEHS), and U.S Food and Drug Administration (FDA) developed a consortium-based research program to link more effectively a variety of hypothesis-based research investigations and guideline-compliant safety testing with BPA. This collaboration is known as the Consortium Linking Academic and Regulatory Insights on BPA Toxicity (CLARITY-BPA). This paper provides a detailed description of the conduct of the study and a midterm update on progress of the CLARITY-BPA research program.

Pharmacokinetics of Melamine and Cyanuric Acid and Their Combinations in F344 Rats

The intentional adulteration of pet food with melamine and cyanuric acid has been implicated in the kidney failure and death of a large number of cats and dogs in the United States. Although individually these compounds present low toxicity in a range of experimental animals, coexposure can lead to the formation of melamine cyanurate crystals in the nephrons and eventual kidney failure. Given this mode of action, a good understanding of the pharmacokinetic profiles of melamine and cyanuric acid and their combinations is essential to define properly the risk associated with different exposure scenarios. Previous studies have investigated the individual pharmacokinetic profiles of melamine and cyanuric acid. In this work, we report a comparison between the pharmacokinetic profiles of melamine and cyanuric acid administered individually, administered simultaneously as the individual compounds, and administered as a preformed melamine cyanurate complex. Although the oral coadministration of 1 mg/kg body weight of melamine and cyanuric acid did not alter significantly the pharmacokinetic profiles in relation to those determined upon individual oral administration of each compound, the administration of equal amounts of each triazine as the preformed melamine cyanurate complex significantly altered the pharmacokinetics, with reduced bioavailability of both compounds, lower observed maximum serum concentrations, delayed peak concentrations, and prolonged elimination half lives. These results indicate that in order to estimate properly the combined nephrotoxic potential of melamine and cyanuric acid, the experimental design of toxicological experiments and the evaluation of animal or human exposure scenarios should consider the detailed mode of exposure, with particular emphasis on any possible ex vivo formation of melamine cyanurate.

A comparison of methylphenidate-, amphetamine-, and methamphetamine-induced hyperthermia and neurotoxicity in male Sprague–Dawley rats during the waking (lights off) cycle

Previous studies focusing on amphetamine (AMPH), methamphetamine (METH) and methylphenidate (MPH) neurotoxicity have almost exclusively been conducted in rodents during the light cycle, which is when most rodents sleep. There are virtually no studies that have simultaneously compared the effects of these three stimulants on body temperature and also determined serum stimulant levels during exposure. The present study compared the effects of MPH, AMPH and METH treatment on body temperature and neurotoxicity during the waking (dark) cycle of the rat. This was done to more effectively replicate stimulant exposure in waking humans and to evaluate the relative risks of the three stimulants when taken inappropriately or non-therapeutically (e.g., abuse). Four subcutaneous injections (4×), at 2 h intervals, were used to administer each dose of the stimulants tested. Several equimolar doses for the three stimulants were chosen to produce plasma levels ranging from 3 times the highest therapeutic levels (no effect on body temperature) to those only attained by accidental overdose or intentional abuse in humans. Either 4×2.0 mg/kg AMPH or 4×2.2 mg/kg METH administered during the waking cycle resulted in peak serum levels of between 1.5 and 2.5 μM (4 to 5 times over maximum therapeutic levels of METH and AMPH) and produced lethal hyperthermia, 70% striatal dopamine depletions, and neurodegeneration in the cortex and thalamus. These results show that METH and AMPH are equipotent at producing lethal hyperthermia and neurotoxicity in laboratory animals during the wake cycle. Administration of either 4×2.2 or 4×3.3 mg/kg METH during the sleep cycle produced lower peak body temperatures, minimal dopamine depletions and little neurodegeneration. These findings indicate that administration of the stimulant during the waking cycle compared to sleep cycle may significantly increase the potency of amphetamines to produce hyperthermia, neurotoxicity and lethality. In contrast, body temperature during the waking cycle was only significantly elevated by MPH at 4×22 mg/kg, and the serum levels producing this effect were 2-fold (approximately 4.5 μM) greater on a molar basis than hyperthermic doses of AMPH and METH. Thus, AMPH and METH were equipotent on a mg/kg body weight basis at producing hyperthermia and neurotoxicity while MPH on a mg/kg body weight basis was approximately 10-fold less potent than AMPH and METH. However, the 10-fold lower potency was in large part due to lower plasma levels produced by MPH compared to either AMPH or METH.