Document G5Oem37jNoadbqrYoNXjokken

AR2-0l?l Kendall B. Wallace, Ph.D., DABT January 27, 2000 Effect of acute FC administration on catalase and acylCoA oxidase expression The following report summarizes the effects of in vivo administration of a single acute dose of N-EtFOSE or PFOS on catalase and acylCoA oxidase gene expression and enzyme activity in liver tissue from exposed rats and guinea pigs. Tissues analyzed in this study were derived from the 3M in-house study "NTBK 116471, pg 100" (a copy of the 3M "Study/Record" is attached) and included approximately 1 g of frozen liver from each of two male and two female rats and guinea pigs that received a single injection of 40 mg/kg PFOS, 160 mg/kg N-Et-FOSE, or an equivalent volume of 2% tween 80 (vehicle control) on March 1, 1999. All animals were killed on March 4, three days following dosing. Portions of the respective livers were quick frozen in liquid nitrogen and shipped on dry ice to Duluth for analysis. Enzyme Sample Preparation - The enzyme fraction consisted of the 6,000 g supernatant of a 10% (wt/vol) homogenate of 0.5-1.0 g frozen liver tissue in 300 mM mannitol-10 mM HEPES-1 mM EGTA (pH 7.2). Protein concentration was estimated according to the method of Bradford using commercial bovine serum albumin as standard. L-CoA Oxidase Assay - The equivalent of ca., 5 pg/ml tissue homogenate was suspended in 60 mM KH2P 0 4-0.02 % Triton X100 (pH 7.4) containing 1 mM p-hydroxyphenylacetate (PHPA), 4 units/ml peroxidase, 20 pM FAD, and 60 pM lauryl-CoA (LCoA). The reactions were allowed to incubate at 37C for 30 min in a shaking water bath and terminated by adding 3 volumes of 2 mM KCN in 100 mM sodium carbonate (pH 10.5). The concentration of H20 2generated during the reaction was estimated from the fluorescence of PHAP as measured with an excitation wavelength of 317 nm and emission at 405 nm. The fluorescence was calibrated with commercial H20 2 and the results are expressed as nmol peroxide generated/min/mg mitochondrial protein (Table 1). Protein was quantitated by the Bradford method. Catalase Assay - The activity of catalase was estimated by a modification of the original method published by Claiborne and Fridovich (J. Biol. Chem. 254, 4245-52, 1979), which is based on the direct measurement of H20 2 disappearance as quantified spectrophotometrically at 240 nm. In this procedure, the tissue sample was diluted in 50 mM potassium phosphate (pH 7.0). The medium was warmed to 27C and the reaction initiated by adding 10.3 mM H20 2. The progress of the reaction was monitored at 240 nm for 5 min. Catalase activity was estimated from the initial linear rate ( E240=43.6 mM^cm'1) and expressed as units/mg protein (Table 1). One unit of activity is defined as that amount of enzyme which catalyzes the decomposition of l pmole of H20 2per min. 004198 i Kendall B. Wallace, Ph.D., DABT January 27, 2000 Northern Blot Analyses - Quantitation of mRNA for both acylCoA oxidase (ACoAO) and catalase were performed by Northern blot analysis of quick frozen liver samples from treated rats and guinea pigs. Approximately 1 g of frozen liver was powderized in liquid nitrogen using a mortar/pestle. Total RNA was recovered using the PERFECT RNATM isolation kit and the concentration quantified spectrophotometrically at 260nm. The RNA was electrophoresed on a 1% agarose gel, blot transferred to a cellulose membrane and hybridized to the corresponding randomly [32P] labeled oligonucleotides that were PCR amplified from primers to ca., 350 base sequence of the respective rat liver gene. mRNA band density was quantified autoradiographically using phospho-imaging software. RESULTS: Bats - Both male and female rats express LCoAO and catalase activities, and there is no substantial difference in enzyme activities between the sexes. The same is true for the constitutive expression of mRNA for both enzymes, with the exception that unexposed male rats do not express the message for PCoAO. Acute exposure to N-Et-FOSE causes a doubling of the specific activity of LCoAO in liver from both male and female rats, but catalase activity is unchanged. Associated with this is a proportionate 2-fold increase in the concentration of mRNA encoding for PCoAO, but no change in catalase mRNA, in livers from both sexes. Again, there is no remarkable sex difference in response to N-Et-FOSE exposure, at-least with this limited number of animals tested (n=2). Exposure of rats to PFOS elicits a similar doubling of LCoAO activity for both sexes, and possibly a slight increase in catalase activity in liver from male rats. Hepatic catalase activity in female rats does not appear to be affected by PFOS exposure. Unlike N-Et-FOSE, which caused a doubling of mRNA band intensity for pCoAO, acute exposure to PFOS caused a 3-6 fold increase in PCoAO mRNA expression. This enhanced expression of message was more pronounced in female compared to male rats, and in both sexes resulted in no greater LCoAO enzyme activity than was observed at half the mRNA level for rats exposed to N-Et-FOSE. Guinea Pigs - The response to guinea pigs to exposure to N-Et-FOSE and PFOS was dramatically different from that observed for rats. The constitutive activity of LCoAO in unexposed guinea pigs of either sex was very low (near LOD values) whereas catalase activity was 1.5-3 times higher. However, the activity of neither enzyme was stimulated following acute exposure to N-Et-FOSE or PFOS in vivo. Perhaps the most dramatic difference between species is the fact that guinea pig mRNA encoding for PCoAO was undetectable, even following exposure to N-Et-FOSE or PFOS. Part of the explanation for this observation may be that we were using hybridization probes developed against the sequence for rat liver ACoAO because the sequence of the guinea pig enzyme is not known at this time. It may be that the guinea pig sequence is sufficiently different that the PCR product lacks complementarity for adequate hybridization to the probe for the rat sequence. Although this may indeed be an artifact, the 004199 2 Kendall B. Wallace, Ph.D., DABT January 27,2000 absence of constitutively expressed message and the inability to stimulate expression of PCoAO mRNA are consistent with the low and uninducible enzyme activity in guinea pigs. CONCLUSIONS: These data provide strong evidence that: 1) N-Et-FOSE and PFOS stimulate both the transcriptional and translational expression of acylCoA oxidase in rats in vivo, and 2) there is a marked difference in the response of rats and guinea pigs to in vivo exposure to these two fluorochemicals. These results are very consistent with the suggestion that these fluorochemical compounds are "peroxisome proliferators" in rats and, much like what has been demonstrated for the classical "peroxisome proliferator" chemicals, guinea pigs are resistant to this effect o f fluorochemical exposures. We were not given the data for organ weights or necropsy findings and thus cannot comment on whether exposure to either agent caused hepatomegaly or other indications that are associated with peroxisome proliferation. However, based on our data, we suspect that the evidence will reveal the classical signs of peroxisome proliferation in rats, but not guinea pigs, caused by these acute exposures. 004200 3 NTBK 116471 dosed 3/1/99 40 mg/kg PFOS sac 3/4/99 160 mg/kg N-Et-FOSE 2% tween 80 Activity___________________________ Animal SPECIES Sfii Is orolein LCoAO Catalase _________ ________ ________ __________ ________ (mg/g) (nmol/min/mq protein) (Units/mq protein) 9R00463 rat M Veh 151.8 4.7 219.9 9R00464 rat M Veh 145.6 5.3 158.5 _______ mRNA oCoAO mRNA (% Band Intensity) 0 0 9R00469 rat 9R00470 rat F F Veh Veh 188.3 153.3 4.7 6.8 141.1 204.7 100 100 9R00465 rat 9R00466 rat M M N-Et-FOSE N-Et-FOSE 157.5 134.9 10.5 11.9 219.5 204.3 290 292 9R00471 rat 9R00472 rat F F N-Et-FOSE N-Et-FOSE 165.8 215.8 10.3 9.3 186.1 171.9 253 320 9R00467 rat 9R00468 rat M M PROS PFC6 115.4 153.0 10.9 13.1 252.0 236.9 482 277 9R00473 rat 9R00474 rat F F PFOS PFOS 143.1 238.6 9.6 11.2 204.1 155.6 630 404 Catalase (% Band Intensity) 127 73 92 108 140 98 118 147 155 116 180 147 9G00045 Gpkt 9G00046 Gpkj M M 9G00051 Gpig 9G00052 Gpig F F 9G00047 Gpiq 9G00048 Gpig 9G00053 Gpig 9G00054 Gpig M M F F 9G00049 Gpig 9G00050 Gpig M M 9G00055 Gpig F 9G00056 GfiS____ F Veh Veh Veh Veh N-Et-FOSE N-Et-FOSE N-Et-FOSE N-Et-FOSE PROS PFOS PFOS PFOS 175.2 251.7 180.3 222.1 212.7 269.1 176.4 218.3 153.2 222.1 127.8 171.6 2.3 1.9 2.6 1.2 3.0 1.4 2.6 1.4 ND 1.0 ND 2.2 314.7 390.7 550.6 488.0 349.3 379.3 416.8 478.1 413.0 460.8 672.9 531.6 0 103 0 97 0 91 0 109 0? 0 92 0 105 0 104 0? 0 97 0 103 0 96 o to Northern Hybridization of Rat Liver Tissue Male Fem ale Methylene Blue Stained Blot Radiograph of P-32 Lableled pCoAO Probe 4 -- -- 290 292 482 277 Cti Ctl 253 320 630 404 B and Intensity (% o f A va ila b le C o ntrols) 0 Z3 0 C/5 i-- 0 > cp Q- CD 0 c D) 'Z3 Q. 0 o c o > CD Ng G) ce0a) o ca 2 -Q V, >, X I* c L_ {ca 0 Methylene Blue Stained Blot PFOS PFOS N-Et-FOSE N-Ei-FOSE Vehicle Vehicle PFOS N-Et-FOSE Vehicle Vehicle N-Et-FOSE Vehicle o Radiograph of P-32 Labeled pCoAO Probe I