Genomic biomarkers of phthalate-induced male reproductive developmental toxicity: a targeted RT-PCR array approach for defining relative potency.

Male rat fetuses exposed to certain phthalate esters (PEs) during sexual differentiation display reproductive tract malformations due to reductions in testosterone (T) production and the expression of steroidogenesis- and INSL3-related genes. In the current study, we used a 96-well real-time PCR array containing key target genes representing sexual determination and differentiation, steroidogenesis, gubernaculum development, and androgen signaling pathways to rank the relative potency of several PEs. We executed dose-response studies with diisobutyl (DIBP), dipentyl (DPeP), dihexyl (DHP), diheptyl (DHeP), diisononyl (DINP), or diisodecyl phthalate (DIDP) and serial dilutions of a mixture of nine phthalates. All phthalates, with the exception of DIDP, reduced fetal testicular T production. Several genes involved in cholesterol transport, androgen synthesis, and Insl3 also were downregulated in a dose-responsive manner by DIBP, DPeP, DHP, DHeP, DINP, and the 9-PE mixture. Despite speculation of peroxisome proliferator activated receptor (PPAR) involvement in the effects of PEs on the fetal testis, no PPAR-related genes were affected in the fetal testes by exposure to any of the tested PEs. Furthermore, the potent PPARα agonist, Wy-14,643, did not reduce fetal testicular T production following gestational day 14-18 exposure, suggesting that the antiandrogenic activity of PEs is not PPARα mediated. The overall sensitivity of the fetal endpoints (gene expression or T production) for the six phthalates from most to least was Cyp11b1 > Star = Scarb1 > Cyp17a1 = T production > Cyp11a1 = Hsd3b = Insl3 > Cyp11b2. The overall potency of the individual phthalates was DPeP > DHP > DIBP ≥ DHeP > DINP. Finally, the observed mixture interaction was adequately modeled by the dose-addition model for most of the affected genes. Together, these data advance our understanding of the collective reproductive toxicity of the PE compounds.

Biomarkers of exposure to triclocarban in urine and serum.

3,4,4'-Trichlorocarbanilide (triclocarban, TCC) is widely used as an antimicrobial agent in a variety of consumer and personal care products. TCC is considered a potential endocrine disruptor, but its potential toxic effects in humans are still largely unknown. Because of its widespread uses, the potential for human exposure to TCC is high. In order to identify adequate exposure biomarkers of TCC, we investigated the metabolic profile of TCC in adult female Sprague Dawley rats after administering TCC once (500 mg/kg body weight) by oral gavage. Urine was collected 0-24 h before dosing, and 0-24 h and 24-48 h after dosing. Serum was collected at necropsy 48 h after dosing. We identified several metabolites of TCC in urine and serum by on-line solid phase extraction-high performance liquid chromatography-mass spectrometry. We unambiguously identified two major oxidative metabolites of TCC, 3'-hydroxy-TCC and 2'-hydroxy-TCC, by comparing their chromatographic behavior and mass spectral fragmentation patterns with those of authentic standards. By contrast, compared to these oxidative metabolites, we detected very low levels of TCC in the urine or serum. Taken together these data suggest that in rats, oxidation of TCC is a major metabolic pathway. We also measured TCC and its oxidative metabolites in 50 urine and 16 serum samples collected from adults in the United States. The results suggest differences in the metabolic profile of TCC in rats and in humans; oxidation appears to be a minor metabolic pathway in humans. Total (free plus conjugated) TCC could serve as a potential biomarker for human exposure to TCC.

Meeting report: moving upstream-evaluating adverse upstream end points for improved risk assessment and decision-making.

BACKGROUND: Assessing adverse effects from environmental chemical exposure is integral to public health policies. Toxicology assays identifying early biological changes from chemical exposure are increasing our ability to evaluate links between early biological disturbances and subsequent overt downstream effects. A workshop was held to consider how the resulting data inform consideration of an "adverse effect" in the context of hazard identification and risk assessment. OBJECTIVES: Our objective here is to review what is known about the relationships between chemical exposure, early biological effects (upstream events), and later overt effects (downstream events) through three case studies (thyroid hormone disruption, antiandrogen effects, immune system disruption) and to consider how to evaluate hazard and risk when early biological effect data are available. DISCUSSION: Each case study presents data on the toxicity pathways linking early biological perturbations with downstream overt effects. Case studies also emphasize several factors that can influence risk of overt disease as a result from early biological perturbations, including background chemical exposures, underlying individual biological processes, and disease susceptibility. Certain effects resulting from exposure during periods of sensitivity may be irreversible. A chemical can act through multiple modes of action, resulting in similar or different overt effects. CONCLUSIONS: For certain classes of early perturbations, sufficient information on the disease process is known, so hazard and quantitative risk assessment can proceed using information on upstream biological perturbations. Upstream data will support improved approaches for considering developmental stage, background exposures, disease status, and other factors important to assessing hazard and risk for the whole population.

Urinary biomarkers of di-isononyl phthalate in rats.

Commercial di-isononyl phthalate (DiNP) is a mixture of various branched-chain dialkyl phthalates mainly containing nine-carbon alkyl isomers. At high doses in rodents, DiNP is a carcinogen, and a developmental toxicant. After exposure, the diester isomers are de-esterified to form hydrolytic monoesters, monoisononyl phthalates (MiNP), which subsequently metabolize to form oxidative metabolites. These metabolites can be excreted in urine or feces. The urinary excretion of DiNP metabolites was monitored in adult female Sprague-Dawley rats after oral administration of a single dose (300 mg/kg) of commercial DiNP. The metabolites were extracted from urine, resolved with high performance liquid chromatography, analyzed by mass spectrometry, and tentatively identified based on their chromatographic separation and mass spectrometric fragmentation pattern. Because DiNP is an isomeric mixture, its metabolites were also isomeric mixtures that eluted from the HPLC column with close retention times. Mono(carboxy-isooctyl)phthalate (MCiOP) was identified as the major metabolite of DiNP; in addition, mono(hydroxy-isononyl)phthalate (MHiNP) and mono(oxo-isononyl)phthalate (MOiNP) were present. Furthermore, metabolites of di-isooctyl phthalate (DiOP) and di-isodecyl phthalate (DiDP) were also detected. Excretion toxicokinetics of the DiNP metabolites in urine followed a biphasic pattern with initial rapid decay in concentration. Despite potential differences in the metabolism of DiNP among species, MCiOP, MHiNP and MOiNP were detected in humans with no known exposure to DiNP at levels significantly higher than MiNP suggesting that these oxidative metabolites may be better urinary biomarkers of human exposure to DiNP than is MiNP.

Urinary metabolites of di-n-octyl phthalate in rats.

Di-n-octyl phthalate (DnOP) is a plasticizer used in polyvinyl chloride plastics, cellulose esters, and polystyrene resins. The metabolism of DnOP results in the hydrolysis of one ester linkage to produce mono-n-octyl phthalate (MnOP), which subsequently metabolizes to form oxidative metabolites. We investigated the toxicokinetics of DnOP in adult female Sprague-Dawley rats by monitoring the excretion of DnOP metabolites in urine after oral administration of DnOP (300 mg/kg). By using authentic standards, the presence of urinary phthalic acid (PA), MnOP, and the major DnOP metabolite, mono-(3-carboxypropyl) phthalate (MCPP) was clearly established. Furthermore, we identified five additional urinary DnOP oxidative metabolites based on their chromatographic behavior and mass spectrometric fragmentation pattern. These DnOP oxidative metabolites, are postulated to be mono-carboxymethyl phthalate (MCMP), mono-(5-carboxy-n-pentyl) phthalate (MCPeP), mono-(7-carboxy-n-heptyl) phthalate (MCHpP), and isomers of mono-hydroxy-n-octyl phthalate (MHOP) (e.g., mono-(7-hydroxy-n-octyl) phthalate) and of mono-oxo-n-octyl phthalate (MOOP) (e.g., mono-(7-oxo-n-octyl) phthalate). The urinary excretion of DnOP metabolites followed a biphasic excretion pattern. The metabolite levels decreased significantly after the first day of DnOP administration although MCPP, MCHpP, MHOP, and MOOP were detectable after 4 days. We also studied the in vitro metabolism of DnOP and MnOP by rat liver microsomes. DnOP produced MnOP, MHOP, and PA in vitro whereas, MnOP produced MHOP and PA in vitro at detectable levels.