MATERIALS AND METHODS

Chemical. PFDA (nonadecafluorodecanoic acid, CAS

No. 335-76-2), 96% purity, was obtained from Aldrich

Chemical Co. (Milwaukee, WI). A stock solution (32.0

mg/ml) was prepared in fresh corn oil (Mazola brand)

and its concentration checked by titration prior to administration.

The solution was stored at 4’C in the dark

and allowed to warm to room temperature before administration.

Animals. All mice used in this study were female.

C57BL/6N (Ahb@) were obtained from Frederick Cancer

Research Center (Frederick, MD). C57BL/6J (Ahbh)

were obtained from Jackson Laboratories (Bar Harbor,

ME). Congenic mice, differing only at the Ah locus, were

obtained from the National Institute of Environmental

Health Sciences (NIEHS) breeding colony, maintained

by backcrossing and intercrossing at the Research Triangle

Institute (Research Triangle Park, NC). The TCDDnonresponsive

congenic mice, C57BL/6J (AhdId), were

originally obtained from Dr. Alan Poland (McArdle Laboratories,

University of Wisconsin) (Bimbaum, 1986).

The responsive heterozygote (AhbId) was obtained by

crossing the nonresponsive congenic mice with the wildtype

responsive C57BL/6J (Ah”/“, strain. The mice were

uniquely identified using numbered ear tags and housed

at the NIEHS under identical room conditions of constant

temperature (22 + I”C), humidity (40-60%), and

lighting (12: 12 lightdark cycle). NIH Open Formula 3 1

and water were provided ad libitum. The four groups

were allowed to acclimate for at least 2 weeks before administration

of the chemical and were 12 to 14 weeks of

age at time of treatment.

Treatment. All mice were randomly assigned to

treatment groups by body weight. Each mouse received a single

dose ranging from 0 to 320 mg PFDA/kg (Table 1) by

gavage at a volume of 10 ml corn oil/kg body wt. The

mice were observed daily for overt signs of toxicity and

body weights were determined three times a week for 30

days. On Day 30 (d30), the surviving mice were weighed

and killed by CO1 asphyxiation. Blood was obtained at

necropsy by cardiac puncture from mice of the C57BL/

6N strain in order to prepare serum for analysis of thyroid

hormones (T, and T4) by radioimmunoassay as previously

described (Brewster et al., 1988). The liver, thymus,

heart, kidney, and spleen were trimmed, weighed,

and fixed in 10% neutral-buffered formalin for light microscopy.

Additional liver sections were stained with

Os04 (Luna, 1968) to determine the presence of fat. Samples

of liver tissue from two mice per group were also

fixed in Fowler’s (2.6% glutaraldehyde-2.0% paraformaldehyde

preparation) fixative (Fowler et al., 1983) for

transmission electron microscopy (TEM). Thin sections

were stained with lead citrate and uranyl acetate.

The C57BL/6N mice received 0, 20, 40, 80, 160, or

320 mg PFDA/kg body wt. The results of studies with

this strain were used to set doses for the studies with the

three congenic strains of mice. The wild-type C57BL/6J

mice were administered 0,40, 80, 100, 120, or 160 mg

PFDA/kg body wt. The responsive heterozygotes, AhbId,

received 0,40,80, or 160 mg PFDA/kg body wt and the

nonresponsive homozygotes, AhdId, received 0, 40, 80,

120, or 160 mg PFDA/kg body wt. In addition, the wildtype

congenic mice [C57BL/6J(Ahb@)J were treated with

0,40, 80, or 120 mg PFDA/kg and were killed at 2, 7,

or 14 days post-treatment to examine the time course of

pathological changes.

Data analyses. When overall significance was found,

dose-response trends were assessed using Jonckheere’s

test. Painvise comparisons were made using the Mann-

Whitney U test. The LD50 estimations were made using

the Spearman-Karber method.

RESULTS

Female C57BL/6J (Ah b/b) 2 day