Mitchell B. Rosen

Toxicogenomic Dissection of the Perfluorooctanoic Acid Transcript Profile in Mouse Liver: Evidence for the Involvement of Nuclear Receptors PPARα and CAR

A number of perfluorinated alkyl acids including perfluorooctanoic acid (PFOA) elicit effects similar to peroxisome proliferator chemicals (PPC) in mouse and rat liver. There is strong evidence that PPC cause many of their effects linked to liver cancer through the nuclear receptor peroxisome proliferator-activated receptor alpha (PPAR alpha). To determine the role of PPAR alpha in mediating PFOA transcriptional events, we compared the transcript profiles of the livers of wild-type or PPAR alpha-null mice exposed to PFOA or the PPAR alpha agonist WY-14,643 (WY). After 7 days of exposure, 85% or 99.7% of the genes altered by PFOA or WY exposure, respectively were dependent on PPAR alpha. The PPAR alpha-independent genes regulated by PFOA included those involved in lipid homeostasis and xenobiotic metabolism. Many of the lipid homeostasis genes including acyl-CoA oxidase (Acox1) were also regulated by WY in a PPAR alpha-dependent manner. The increased expression of these genes in PPAR alpha-null mice may be partly due to increases in PPAR gamma expression upon PFOA exposure. Many of the identified xenobiotic metabolism genes are known to be under control of the nuclear receptor CAR (constitutive activated/androstane receptor) and the transcription factor Nrf2 (nuclear factor erythroid 2-related factor 2). There was excellent correlation between the transcript profile of PPAR alpha-independent PFOA genes and those of activators of CAR including phenobarbital and 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) but not those regulated by the Nrf2 activator, dithiol-3-thione. These results indicate that PFOA alters most genes in wild-type mouse liver through PPAR alpha, but that a subset of genes are regulated by CAR and possibly PPAR gamma in the PPAR alpha-null mouse.

Gene Profiling in the Livers of Wild-type and PPARα-Null Mice Exposed to Perfluorooctanoic Acid

Health concerns have been raised because perfluorooctanoic acid (PFOA) is commonly found in the environment and can be detected in humans. In rodents, PFOA is a carcinogen and a developmental toxicant. PFOA is a peroxisome proliferator-activated receptor α (PPARα) activator; however, PFOA is capable of inducing heptomegaly in the PPARα-null mouse. To study the mechanism associated with PFOA toxicity, wild-type and PPARα-null mice were orally dosed for 7 days with PFOA (1 or 3 mg/kg) or the PPARα agonist Wy14,643 (50 mg/kg). Gene expression was evaluated using commercial microarrays. In wild-type mice, PFOA and Wy14,643 induced changes consistent with activation of PPARα. PFOA-treated wild-type mice deviated from Wy14,643-exposed mice with respect to genes involved in xenobiotic metabolism. In PFOA-treated null mice, changes were observed in transcripts related to fatty acid metabolism, inflammation, xenobiotic metabolism, and cell cycle regulation. Hence, a component of the PFOA response was found to be independent of PPARα. Although the signaling pathways responsible for these effects are not readily apparent, overlapping gene regulation by additional PPAR isoforms could account for changes related to fatty acid metabolism and inflammation, whereas regulation of xenobiotic metabolizing genes is suggestive of constitutive androstane receptor activation.

Comparative Hepatic Effects of Perfluorooctanoic Acid and WY 14,643 in PPAR-α Knockout and Wild-type Mice

Perfluorooctanoic acid (PFOA) is a chemical used in the production of fluoropolymers. Its persistence in the environment and presence in humans and wildlife has raised health concerns. Liver tumor induction by PFOA is thought to be mediated in rodents by PPAR-α. A recent US EPA scientific advisory board questioned the contribution of PPAR-α in PFOA-induced liver tumors. Liver response in CD-1, SV/129 wild-type (WT), and PPAR-α knockout (KO) SV/129 mice was evaluated after seven daily treatments of PFOA-NH4 + (1, 3, or 10 mg/kg, p.o.) or the prototype PPARα-agonist Wyeth 14,643 (WY, 50 mg/kg). Livers were examined by light and electron microscopy. Proliferation was quantified after PCNA immunostaining. PFOA treatment induced a dose-dependent increase in hepatocyte hypertrophy and labeling index (LI) similar to WY in WT mice. Ultrastructural alterations of peroxisome proliferation were similar between WY-treated and 10 mg/kg PFOA-treated WT mice. KO mice had a dose-dependent increase in hepatocyte vacuolation but increased LI only at 10 mg PFOA/kg. WY-treated KO mice were not different from KO control. These data suggest that PPAR-α is required for WY- and PFOA-induced cellular alterations in WT mouse liver. Hepatic enlargement observed in KO mice may be due to an accumulation of cytoplasmic vacuoles that contain PFOA.

Developmental effects of maternal stress in the CD-1 mouse induced by restraint on single days during the period of major organogenesis

Maternal stress during gestation can produce significant fetal and/or postnatal effects, and can enhance the teratogenicity of other agents. We have previously shown that restraint stress on gestational day 8 in CD-1 mice produces significant increases in encephaloceles and supernumerary and fused ribs. In the present study we have examined the effects of stress induced by restraint on individual days during the period of major organogenesis (days 6-14). Weight loss and stress-induced analgesia as assessed by the tail-flick method were used to determine the degree of stress induced by a 12-h restraint period. Restrained animals lost significantly more weight and had longer tail-flick latencies than the concurrent food and water deprived controls on all gestational days. Significant increases in embryo/fetal mortality were also observed in the offspring of restrained animals. An increased incidence of supernumerary ribs was found in mice restrained on days 7 and 8. Since maternal toxicity induced by chemical teratogens may be accompanied by a general increase in maternal stress, our data suggest that such stress may be an etiological factor in teratology bioassays in which dose levels are sufficiently high to induce overt maternal toxicity.

Does Exposure to Perfluoroalkyl Acids Present a Risk to Human Health

The information in this document has been funded by the U.S. Environmental Protection Agency. It has been subjected to review by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents reflect the views of the Agency or does mention of trade names or commercial products constitute endorsement or recommendation for use.

Toxicity and recovery in the pregnant mouse after gestational exposure to the cyanobacterial toxin, cylindrospermopsin

Cylindrospermopsin (CYN) is a tricyclic alkaloid toxin produced by fresh water cyanobacterial species worldwide. CYN has been responsible for both livestock and human poisoning after oral exposure. This study investigated the toxicity of CYN to pregnant mice exposed during different segments of gestation. The course of recovery and individual responses to the toxin were evaluated. Adverse effects of CYN were monitored up to 7 weeks post‐dosing by clinical examination, histopathology, biochemistry and gene expression. Exposure on gestational days (GD) 8–12 induced significantly more lethality than GD13–17 exposure. Periorbital, gastrointestinal and distal tail hemorrhages were seen in both groups. Serum markers indicative of hepatic injury (alanine amino transferase, aspartate amino transferase and sorbitol dehydrogenase) were increased in both groups; markers of renal dysfunction (blood urea nitrogen and creatinine) were elevated in the GD8–12 animals. Histopathology was observed in the liver (centrilobular necrosis) and kidney (interstitial inflammation) in groups exhibiting abnormal serum markers. The expression profiles of genes involved in ribosomal biogenesis, xenobiotic and lipid metabolism, inflammatory response and oxidative stress were altered 24 h after the final dose. One week after dosing, gross, histological and serum parameters had returned to normal, although increased liver/body weight ratio and one instance of gastrointestinal bleeding was found in the GD13–17 group. Gene expression changes persisted up to 2 weeks post‐dosing and returned to normal by 4 weeks. Responses of individual animals to CYN exposure indicated highly significant inter‐animal variability within the treated groups. Copyright

Postnatal evaluation of prenatal exposure to p-xylene in the rat

Pregnant Sprague-Dawley rats were exposed to either 3500 or 7000 mg/m3 p-xylene from days 7-16 of gestation. Dams were allowed to give birth, and litters were counted, weighed, and observed for external malformations on postnatal days (PD) 1 and 3. Litters were normalized to 8 pups (4 males and 4 females +/- 1) on PD4. On PD21 animals were weaned and littermates housed by sex. Body weights were recorded weekly until weaning and once every 2 weeks thereafter. Central nervous system (CNS) development was evaluated by acoustic startle response on PD13, 17, 21, and 63 as well as figure-8 maze activity on PD22 and 65. Maternal weight gain during the treatment period was significantly less in the high-dose group. No effects were seen on litter size or weight at birth or on PD3. There were no effects of xylene exposure on growth rate. There were no treatment-related effects on acoustic startle response or figure-8 maze activity. Thus, p-xylene as administered in this study does not appear to be a selective developmental toxicant in the rat.

Developmental toxicity of perfluorononanoic acid in mice

Perfluorononanoic acid (PFNA) is a ubiquitous and persistent environmental contaminant. Although its levels in the environment and in humans are lower than those of perfluorooctane sulfonate (PFOS) or perfluorooctanoic acid (PFOA), a steady trend of increases in the general population in recent years has drawn considerable interest and concern. Previous studies with PFOS and PFOA have indicated developmental toxicity in laboratory rodent models. The current study extends the evaluation of these adverse outcomes to PFNA in mice. PFNA was given to timed-pregnant CD-1 mice by oral gavage daily on gestational day 1-17 at 1, 3, 5 or 10mg/kg; controls received water vehicle. Dams given 10mg/kg PFNA could not carry their pregnancy successfully and effects of this dose group were not followed. Similar to PFOS and PFOA, PFNA at 5mg/kg or lower doses produced hepatomegaly in the pregnant dams, but did not affect the number of implantations, fetal viability, or fetal weight. Mouse pups were born alive and postnatal survival in the 1 and 3mg/kg PFNA groups was not different from that in controls. In contrast, although most of the pups were also born alive in the 5mg/kg PFNA group, 80% of these neonates died in the first 10 days of life. The pattern of PFNA-induced neonatal death differed somewhat from those elicited by PFOS or PFOA. A majority of the PFNA-exposed pups survived a few days longer after birth than those exposed to PFOS or PFOA, which typically died within the first 2 days of postnatal life. Surviving neonates exposed to PFNA exhibited dose-dependent delays in eye opening and onset of puberty. In addition, increased liver weight seen in PFNA-exposed offspring persisted into adulthood and was likely related to the persistence of the chemical in the tissue. Evaluation of gene expression in fetal and neonatal livers revealed robust activation of peroxisome proliferator-activated receptor-alpha (PPARα) target genes by PFNA that resembled the responses of PFOA. Our results indicate that developmental toxicity of PFNA in mice is comparable to that of PFOS and PFOA, and that these adverse effects are likely common to perfluoroalkyl acids that persist in the body.

Subtractive hybridization: A technique for the isolation of differentially expressed genes

The technique of subtractive hybridization has been successfully used to isolate and identify rare and/or differentially expressed genes in specific tissues or under specific conditions. This technique allows for the isolation and subsequent identification of cDNA clones that can represent as little as 0.01% of the original mRNA population. Alterations in gene expression are invariably closely related to the primary initiation of changes in normal embryonic development, if not the primary event itself. Similarly, manifestations of cellular/tissue toxicity will be reflected in changes in gene expression of the target sites. The ability to isolate changing gene species, and subsequently to characterize the protein(s) they form, means that subtractive hybridization is potentially a powerful tool for characterizing both normal and abnormal conditions in animals and plants. This report reviews some of the recent applications of this technique, details a protocol we have used successfully in studies on abnormal...

In Situ Hybridization and Oligomer Probes: Evaluation of Gene Expression During Development

Alteration of gene expression can result in numerous pathologies, including proliferative diseases, functional deficits, and developmental defects. Recent studies have suggested that changes in the expression domains of Homeotic (Hox) genes during development are associated with the induction of chemically induced developmental anomalies. Before meaningful data on the alteration of gene expression can be obtained, the heterogeneity of normal expressions within a species must be evaluated. In situ hybridization is a powerful technique that can detect perturbations in the spatial and/or temporal expression of genes. The hybridization protocol described here utilizes 33P end-labeled, single-stranded DNA oligomeric nucleotides. Some of the technical advantages to this approach are that (1) DNA probes are not sensitive to RNase, (2) the probes are based on published sequences and are chemically synthesized, (3) probes are end-labeled utilizing a terminal transferase reaction, producing probes of high specific ...