Naomi Kudo

Sex hormone-regulated renal transport of perfluorooctanoic acid

The biological half-life (t1/2) of perfluorooctanoic acid (PFOA) in male rats is 70 times longer than that in female rats. The difference is mainly due to the difference in renal clearance (CL(R)), which was significantly reduced by probenecid, suggesting that PFOA is excreted by organic anion transporter(s). Castration of male rats caused a 14-fold increase in the CL(R) of PFOA, which made it comparable with that of female rats. The elevated PFOA CL(R) in castrated males was reduced by treating them with testosterone. Treatment of male rats with estradiol increased the CL(R) of PFOA. In female rats, ovariectomy caused a significant increase in CL(R) of PFOA, which was reduced by estradiol treatment. Treatments of female rats with testosterone reduced the CL(R) of PFOA as observed in castrated male rats. To identify the transporter molecules that are responsible for PFOA transport in rat kidney, renal mRNA levels of organic anion transporter 1 (OAT1), OAT2, OAT3, organic anion transporting polypeptide 1 (oatp1), oatp2 and kidney specific organic anion transporter (OAT-K) were determined in male and female rats under various hormonal states and compared with the CL(R) of PFOA. The level of OAT2 mRNA in male rats was only 13% that in female rats. Castration or estradiol treatment increased the level of OAT2 mRNA whereas treatment of castrated male rats with testosterone reduced it. In contrast to OAT2, mRNA levels of both oatp1 and OAT-K were significantly higher in male rats compared with female rats. Castration or estradiol treatment caused a reduction in the levels of mRNA of oatp1 and OAT-K in male rats. Ovariectomy of female rats significantly increased the level of OAT3 mRNA. Multiple regression analysis suggests that the change in the CL(R) of PFOA is, at least in part, due to altered expression of OAT2 and OAT3.

Comparison of the toxicokinetics between perfluorocarboxylic acids with different carbon chain length.

Toxicokinetics was compared between perfluoroheptanoic acid (PFHA), perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) in male and female rats. Half lives (t(1/2)) in male and female rats were calculated to be 0.10 and 0.05 days, respectively, for PFHA, 5.63 and 0.08 days for PFOA, 29.5 and 2.44 days for PFNA and 39.9 and 58.6 days for PFDA. Total clearance (CL(tot)) of PFHA was higher than those of other perfluorocarboxylic acids (PFCAs) in both male and female rats. By contrast, CL(tot) of PFDA was extremely low in both sexes. PFCAs having shorter carbon chain length showed higher CL(tot). There was a significant sex-related difference in CL(tot) of PFOA and PFNA. Distribution volumes in steady state (V(ss)) were not much different between PFCAs and between sexes. To estimate the role of urinary excretion in plasma clearance of PFCA, renal clearance (CL(R)) was determined for PFCAs. CL(R) of PFCAs were in the order PFHA>PFOA>PFNA approximately equal PFDA and PFHA approximately equal PFOA>PDNA>PFDA in male and female rats, respectively. There was a close relationship between CL(tot) and CL(R) (r(2)=0.981). Plasma protein binding, estimated in vitro, was over 98% for all PFCAs tested. The results indicate that CL(R) is responsible for the difference in CL(tot) between PFCAs having different carbon chain length and between sexes.

Comparison of the elimination between perfluorinated fatty acids with different carbon chain length in rats.

Elimination in urine and feces was compared between four perfluorinated fatty acids (PFCAs) with different carbon chain length. In male rats, perfluoroheptanoic acid (PFHA) was rapidly eliminated in urine with the proportion of 92% of the dose being eliminated within 120 h after an intraperitoneal injection. Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) was eliminated in urine with the proportions of 55, 2.0 and 0.2% of the dose, respectively. By contrast, four PFCAs were eliminated in feces with the proportion of less than 5% of the dose within 120 h after an injection. In female rats, the proportions of PFOA and PFNA eliminated in urine within 120 h were 80% and 51% of the dose, respectively, which were significantly higher compared with those in male rats. There was the tendency that PFCA with longer carbon chain length is less eliminated in urine in both male and female rats. Fecal elimination of PFCAs was not different between PFCAs in female rats and comparable to those in male rats. The rates of biliary excretion of PFCAs in male rats were slower than those in female rats. Sex-related difference in urinary elimination of PFOA was abolished when male rats had been castrated. On the contrary, treatment with testosterone suppressed the elimination of PFOA in urine in both castrated male rats and female rats. The effect of testosterone was in a time- and dose-dependent manner. These results suggest that PFCAs are distinguished by their carbon chain length by a renal excretion system, which is regulated by testosterone.

Induction by perfluorinated fatty acids with different carbon chain length of peroxisomal β-oxidation in the liver of rats

The potency of the induction of peroxisomal beta-oxidation was compared between perfluorinated fatty acids (PFCAs) with different carbon chain lengths in the liver of male and female rats. In male rats, perfluoroheptanoic acid (PFHA) has little effect, although perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA) and perfluorodecanoic acid (PFDA) potentially induced the activity. By contrast, PFHA and PFOA did not induce the activity of peroxisomal beta-oxidation in the liver of female rats while PFNA and PFDA effectively induced the activity. The induction of the activity by these PFCAs was in a dose-dependent manner, and there is a highly significant correlation between the induction and hepatic concentrations of PFCAs in the liver regardless of their carbon chain lengths. These results strongly suggest that the difference in their chemical structure is not the cause of the difference in the potency of the induction. Hepatic concentrations of PFOA and PFNA was markedly higher in male compared with female rats. Castration of male rats reduced the concentration of PFNA in the liver and treatment with testosterone entirely restored the reduction. In contrast to the results obtained from the in vivo experiments, the activity of peroxisomal beta-oxidation was induced by PFDA and PFOA to the same extent in cultured hepatocytes prepared from both male and female rats. These results, taken together, indicate that difference in accumulation between PFCAs in the liver was responsible for the different potency of the induction of peroxisomal beta-oxidation between PFCAs with different carbon chain lengths and between sexes.

Determination of perfluorinated carboxylic acids in biological samples by high-performance liquid chromatography

This paper describes a method for the quantitative determination of perfluorinated carboxylic acids (PFCAs), perfluorohexanoic acid (C6-PFCA), perfluoroheptanoic acid (C7-PFCA), perfluorooctanoic acid (C8-PFCA), perfluorononanoic acid (C9-PFCA) and perfluorodecanoic acid (C10-PFCA), in biological samples. PFCA in liver homogenates was extracted as an ion pair with tetrabutylammonium (TBA) ion into organic solvent, then the PFCA was derivatized with 3-bromoacetyl-7-methoxycoumarin (BrAMC) and quantified by HPLC with fluorescence detection. This method is applicable for the studies on tissue accumulation and elimination of PFCAs in animals after the administration.

Alterations by perfluorooctanoic acid of glycerolipid metabolism in rat liver.

The effects of perfluorooctanoic acid (PFOA) feeding on hepatic levels of glycerolipids and the underlying mechanism were investigated. Feeding of rats with 0.01% of PFOA in the diet for 1 week caused an increase in the contents of phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer) and triglyceride (TG), which were 2.2, 2.4, 2.4, 1.6 and 5.2 times over control, respectively, on the basis of whole liver. The activities of glycerol-3-phosphate acyltransferase, diacylglycerol kinase and PtdSer decarboxylase were significantly increased upon PFOA feeding, whereas the activities of CTP:phosphoethanolamine cytidylyltransferase and PtdEtn N-methyltransferase were decreased. On the other hand, the activity of CTP:phosphocholine cytidylyltransferase was not increased by PFOA. Upon PFOA feeding, hepatic level of 16:0-18:1 PtdCho was markedly increased and, by contrast, the levels of molecular species of PtdCho which contain 18:2 were decreased, resulting in the reduced concentration of molecular species of serum PtdCho containing 18:2. The increase in the level of hepatic 16:0-18:1 PtdCho seemed to be due to 3-fold increase in the activities of both delta9 desaturase and 1-acylglycerophosphocholine (1-acyl-GPC) acyltransferase. The mechanism by which PFOA causes the accumulation of glycerolipids in liver was discussed.

Determination of perfluorocarboxylic acids by gas-liquid chromatography in rat tissues

A method for the determination of tissue perfluorocarboxylic acids (PFCA) was developed using gas-liquid chromatography with an electron capture detector (ECD). Perfluorooctanoic acid (PFOA), perfluorononanoic acid (PFNA), and perfluorodecanoic acid (PFDA) were efficiently extracted from rat liver, methylated with diazomethane, and separated on GLC. Internal standards that were added to liver homogenates were used in the quantitative analysis of PFCAs to correct the loss during the extraction and derivatization. The concentration of PFDA in rat liver 24 h after intraperitoneal administration at a dose of 20 mg/kg body weight was 113.9 +/- 11.4 microg/g liver. The value corresponds to the previously reported results that were obtained using [14C]PFDA.

Regulation by carbohydrate and clofibric acid of palmitoyl-CoA chain elongation in the liver of rats

Regulation of palmitoyl-CoA chain elongation (PCE) and its contribution to oleic acid formation were investigated in rat liver in comparison with stearoyl-CoA desaturase (SCD). Hepatic PCE activity was induced by the administration of 20% wt/vol glucose or fructose in the drinking water of normal rats. In streptozotocin-induced diabetic rats, the activities of both PCE and SCD were suppressed, and fructose, but not glucose, feeding caused an increase in the activities of both enzymes. Treatment of normal rats with clofibric acid in combination with carbohydrate further increased PCE, but not SCD, acitivity. FA analysis of hepatic lipids revealed that the proportion of oleic acid (18∶1n−9) increased upon administration of carbohydrate or clofibric acid. The treatment of rats with clofibric acid in combination with carbohydrate greatly increased the proportion of 18∶1n−9. A significant correlation was observed between PCE activity and the hepatic proportion of 18∶1n−9 (r2=0.874, Pº0.01), whereas the relationship between SCD activity and the proportion of 18∶1n−9 was not significant (r2=0.552, P>0.05). Taken together, these results suggest that carbohydrate induces PCE as well as SCD activity to increase the hepatic 18∶1 content in rat liver, and the increased PCE activity seems to be responsible for the further increase in 18∶1n−9 when carbohydrate is administered in combination with clofibric acid.

Up-Regulation of Stearoyl-CoA Desaturase 1 Increases Liver MUFA Content in Obese Zucker but Not Goto-Kakizaki Rats

The Goto-Kakizaki (GK) rat is an animal model for spontaneous-onset, non-obese type 2 diabetes. Despite abundant evidence about disorders in metabolism, little information is available about fatty acid metabolism in the liver of GK rats. This study aimed to investigate the characteristics of the fatty acid profile, particularly MUFA, and the mechanism underlying the alterations in fatty acid profiles in the liver of GK rats. The activities of enzymes that participate in the biosynthesis of MUFA, expressions of genes encoding these enzymes, and the fatty acid profile in the liver were compared with those of obese Zucker (fa/fa) (ZF) rats, which are obese and non-diabetic. Stearoyl-CoA desaturase (SCD) activity and SCD1 gene expression were considerably up-regulated in GK rats, and these levels were largely comparable to those in ZF rats. However, the proportions and contents of oleic acid and palmitoleic acid were very low considering the highly elevated activity of SCD in the liver of GK rats, when compared with ZF rats. Palmitoyl-CoA chain elongation (PCE) activity and fatty acid elongase (Elovl6) gene expression were markedly up-regulated in ZF rats, whereas PCE activity was up-regulated much less and Elovl6 gene expression was unchanged in GK rats. These results suggest the possibility that up-regulation of gene expression of Elovl6 along with SCD1 is indispensable to elevate the proportions and contents of oleic acid in the liver.

Metabolism and Pharmacokinetics

Perfluoroalkyl acids (PFAAs) are highly persistent and widely spread in the environment. PFAAs were detected in various wildlife and human after 1960s and the levels gradually elevated to 2000. In addition to the production of perfluorocarboxylic acids (PFCAs) themselves, fluorotelomer-based compounds were potential source of PFCAs. Fluorotelomer-based compounds can degrade through atmospheric oxidation and biodegradation to form PFCAs. The biological half-lives (t1/2) of perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS), major contaminants in the environment, were calculated to be 3.5 and 8.5 years in human, respectively. To elucidate the mechanisms by which PFAAs accumulate in human, pharmacokinetics have been studied in experimental animals, however, in rats, mice, monkeys and other animals, half-life (T1/2) were hours to days, therefore, great species-difference exist in t1/2 between experimental animals and human. Recent studies identified partially the biological molecules responsible for protein binding, transmembrane transport of PFCAs. In addition, transplacental and lactational transports are thought to be an important exposure routes of these chemicals, because developmental toxicity of PFAAs is thought to be one of primary toxic events of PFAAs. Physiologically-based pharmacokinetic (PBPK) models are proposed to understanding kinetics of PFAAs in biological systems.