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(The FASEB Journal. 2002;16:1001-1009.)
© 2002 FASEB

Embryonic prostaglandin H synthase-2 (PHS-2) expression and benzo[a]pyrene teratogenicity in PHS-2 knockout mice1

TOUFAN PARMAN* and PETER G. WELLS*,{dagger}2

* Faculty of Pharmacy and
{dagger} Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5S 2S2

2Correspondence: Peter G. Wells, Faculty of Pharmacy, University of Toronto, 19 Russell Street, Toronto, Ontario, Canada M5S 2S2.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The developmental role of prostaglandin H synthase-2 (PHS-2), which converts xenobiotics such as benzo[a]pyrene (B[a]P) to toxic free radical intermediates, is poorly understood. In this study, we determined the embryonic expression and teratological relevance of PHS-2 in pregnant CD-1 and B6/129S7 PHS-2 knockout mice. Wild-type (+/+) B6/129S7 dams given B[a]P on gestational day (GD) 10 had three times more fetal malformations than did +/- PHS-2-deficient dams (P<0.05). GD 10–13 CD-1 embryos had high PHS-2 protein expression, and both + /+ and +/- GD 19 B6/129S7 fetuses had more B[a]P-initiated malformations and postpartum lethality than did -/- littermates (P<0.05). Thus, embryonic PHS-2 is expressed constitutively during organogenesis and contributes substantially to B[a]P teratogenicity.—Parman, T., Wells, P. G. Embryonic prostaglandin H synthase-2 (PHS-2) expression and benzo[a]pyrene teratogenicity in PHS-2 knockout mice.


Key Words: cyclooxygenase-2 • free radicals • reactive oxygen species • developmental biology • toxicology


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PROSTAGLANDIN H SYNTHASE (PHS), often referred to by its cyclooxygenase (COX) component, exists as two isoforms, PHS-1 (COX-1) and PHS-2 (COX-2), and catalyzes initial steps in the biosynthesis of prostaglandins and thromboxanes (1) . PHS-1 is expressed constitutively in most adult tissues and is available for on-demand synthesis of prostaglandins that participate in "housekeeping" activities, such as regulating vascular homeostasis, stomach function, and renal water and sodium resorption (2 3 4) . In contrast, PHS-2, which is involved in inflammatory responses, is generally nonconstitutive but is inducible in adult tissues, except for the macula densa of the kidney (5) , vas deferens (6) , and brain (7) , where it is constitutively expressed. PHS-2 expression has not been investigated in embryonic tissues during the teratologically susceptible period of organogenesis.

Benzo[a]pyrene (B[a]P), a polycyclic aromatic hydrocarbon formed from the incomplete combustion of organic matter, is a carcinogen in both humans and animals (8) and is a teratogen in animals (9 , 10) . The mutagenicity, carcinogenicity, and teratogenicity of B[a]P are thought to depend at least in part on its enzymatic bioactivation to electrophilic and/or free radical reactive intermediates, and the subsequent irreversible damage to macromolecular targets (10 11 12) . B[a]P bioactivation is catalyzed by several enzymes, including cytochromes P450 (P450, CYP) (8) , in particular P4501A1 (CYP1A1), and peroxidases such as PHS (1 , 13) . In the latter case, during the conversion of prostaglandin G2 to prostaglandin H2 by the hydroperoxidase component of PHS, xenobiotics can serve as reducing cosubstrates, themselves being oxidized to reactive free radical intermediates. If not detoxified, these reactive intermediates can oxidize and/or arylate DNA and proteins (11 , 14 15 16 17) . Arylation of embryonic DNA and protein by an electrophilic reactive intermediate has been implicated in the mechanism of B[a]P teratogenicity (9) . DNA oxidation may also constitute an important molecular mechanism mediating teratogenicity, because B[a]P causes embryonic DNA oxidation, and both this macromolecular damage and the embryopathic effects of B[a]P in embryo culture are blocked by the antioxidative enzyme catalase (18) . Furthermore, the teratogenicity of B[a]P is enhanced in p53-deficient knockout mice, which are deficient in DNA repair, which corroborates the teratological importance of DNA damage (10) .

Although PHSs are involved in bioactivation of xenobiotics (1 , 13) , including proteratogens (12 , 19) , to toxic free radical reactive intermediates, the relative in vivo contributions of the two PHS isozymes are unknown. More particularly, nothing is known about the expression of embryonic PHS-2, let alone its role in proteratogen bioactivation, during organogenesis, the period of susceptibility to teratogens. The recent availability of PHS-2 knockout mice (2 , 3) permitted the first direct assessment of the dependence of B[a]P teratogenicity on PHS-2-catalyzed embryonic bioactivation. The potential role of PHS-2-catalyzed bioactivation was corroborated by the first demonstration in wild-type littermates of strong constitutive expression of embryonic PHS-2 protein during organogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals
B[a]P, ethidium bromide, glycine, Tween 20, Tris, sodium diethyldithiocarbamate (DDC), mannitol, and EDTA were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). Bis-acrylamide solution (30%) and xylene cyanol were obtained from Bio-Rad Laboratories (Richmond, CA). Ficoll type 400 was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Agarose gel, sodium dodecyl sulfate, ammonium persulfate, N,N,N', N'-tetramethylethylenediamine (TEMED), and mercaptoethanol came from ICN Biomedical (Aurora, OH). The primers were purchased from the Center for Applied Genomics at the Hospital for Sick Children (Toronto, ON, Canada). Taq polymerase and dNTPs were purchased from Perkin Elmer Canada (Mississauga, ON, Canada). Phenylmethylsulfonyl fluoride (PMSF) was purchased from Boehringer Mannheim (Indianapolis, IN). The PHS-2 protein standard was from Cayman Chemicals (Ann Arbor, MI). Polyclonal anti-PHS-2 raised in rabbits was from Oxford Biomedical Research (Oxford, MO). Horseradish peroxidase-conjugated anti-rabbit IgG was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). DNA ladder and protein markers, broad range (premixed format), were obtained from New England Biolabs (Mississauga, ON, Canada).

Teratogenesis
Breeding pairs of B6;129S-Ptgs2tmIJed mice (referred to herein as B6/129S7, or PHS-2 knockouts) were purchased from Jackson Laboratories (Stock No. 002476, Bar Harbor, ME). A breeding colony was established, wherein animals were housed in microisolator cages and cage bedding was changed in a laminar flow hood to maintain a pathogen-free status. Because the homozygous PHS-2-deficient females are not fertile, only the wild-type and heterozygous (+/-) females were housed overnight three to a cage with a heterozygous male breeder. All PHS-2 genotypes were confirmed in our laboratory. The presence of a vaginal plug the next morning was designated as gestational day (GD) 1. Pregnant females were isolated and provided with food (Rodent Chow, Ralston Purina, St. Louis, MO) and tap water ad libitum, and a 12- h light-dark cycle was maintained automatically. Females were treated with either B[a]P (200 mg/kg i.p.) or corn oil vehicle at 8:00 AM on GD 10, the time of teratological susceptibility (10) .

On GD 19, females were killed by cervical dislocation. The uterine horns were exteriorized, the number and location of implantation sites (fetuses and resorptions) were noted, the fetuses were removed, and viable fetuses were placed under a heat lamp at 30°C for 2 h to determine postpartum survival. At the end of this period, fetuses were weighed and examined for gross anomalies that are listed in Fig. 5 . Resorptions were dissected free from the uterus, flash frozen in liquid nitrogen, and stored at -80°C for PHS-2 genotyping accomplished by using polymerase chain reaction (PCR).



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  		Figure 5. Effect of embryonic genotype on B[a]P teratogenicity. Fetuses are combined from +/- PHS-2-deficient and + /+ PHS-2-normal dams. The n indicates the number of fetuses born alive. The asterisk (P<0.01) and cross (P=0.07) indicate the level of significance for the respective differences from the -/- PHS-2 knockout fetuses.

Genotyping
Resorptions and tail snips from dams, as well as the fetal tails, were obtained, and DNA was isolated from each sample by using a QIAGEN DNA extraction kit (QIAGEN, Chatworth, CA). The DNA concentration was determined spectrophotometrically at 260 nm (Model Lambda 3, Perkin Elmer Canada). Samples for DNA amplification were prepared by adding 800 ng of DNA to PCR master mix containing PCR 1x buffer, 0.2 mM dNTPs, 2.5 mM MgCl2, and either primer to amplify a 200- bp band for the neocassette: OIMR013 (5'- CTT GGG TGG AGA GGC TAT TC-3') (Neo, sense), 1 µM; and OIMR014 (5'-AGG TGA GAT GAC AGG AGA TC-3') (neo, antisense), 1 µM; or primers to amplify a 900- bp band for the PHS-2 gene: OIMR546 (5'-ATC TCA GCA CTG CAT CCT GC-3') (mouse PHS-2 exon 1, sense), 1 µM; and OIMR547 (5'-CAC CAT AGA ATC CAG TCC GG-3') (mouse exon 2, antisense), 1 µM; double-distilled water (ddH2O) and Taq polymerase (1.25 U), for a final reaction volume of 50 µl. Sequences for the primers were kindly provided by the Jackson Laboratory. Samples were placed in a thermal cycler (Perkin Elmer 2400, Perkin Elmer Canada) and run under the following conditions: 94°C, 3 min; 94°C, 45 s; 59°C, 1 min; 72°C, 3 min for 35 cycles and a final extension step of 72°C, 2 min. Added to the program was a 4°C "forever" final step to ensure that samples would be kept at 4°C once the PCR was complete until samples would be examined. PCR samples were combined with 6x gel loading buffer (0.25% bromphenol blue, 0.25% xylene cyanol, and 15% Ficoll type 400 in ddH2O) and loaded onto a 1% agarose gel. Gels were run at a constant 100 V for 1 h, stained with 1x Tris/borate/EDTA containing ethidium bromide (150 µg/ml) for 15–20 min, washed twice, and photographed.

Immunoblotting
Untreated pregnant CD-1 dams (Charles River Canada, St. Constant, Quebec, Canada) were killed by cervical dislocation on GDs 10–13, which encompasses the period of organogenesis for mice. Maternal organs and embryos with yolk sacs were flash frozen in liquid nitrogen. Microsomes were prepared from maternal tissues and embryos by using a modified method of Johnson et al. (20) . Briefly, partially thawed embryos (15 embryos pooled) or tissues (four of each tissue per tube) were homogenized in 0.1 M phosphate buffer, pH 7.8, containing 10 mM EDTA, 250 mM mannitol, and 300 µM DDC. The crude homogenate was centrifuged at 10,000 g for 20 min at 4°C. The S-9 supernatant was centrifuged at 100,000 g for 2 h to isolate the microsomes. Subsequently, the microsomal protein was solubilized for 45 min with 1% Tween 20 in 80 mM Tris-HCl, pH 8.0, containing 300 µM DDC, 1 mM PMSF, and 500 µM EDTA . The protein was measured by using a DC Protein Assay Protocol (Bio-Rad) according to the manufacturer’s instructions. The entire procedure was performed at 0–4°C. Proteins were separated electrophoretically under reducing conditions, and the bands were detected with an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech).

Statistical analysis
Continuous data were analyzed by one-way ANOVA or Student’s t test as appropriate. Binomial data were analyzed by {chi}2 analysis or the Fisher exact test as appropriate. A P value of less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Embryonic death and teratogenesis
Characterization of the B6/129S7 model: B[a]P vs. vehicle-treated controls
Analysis by maternal genotype
Susceptibility of the B6/129S7 hybrid strain to B[a]P has not been previously determined, so this strain was first characterized for B[a]P teratogenicity by using PHS-2 +/- dams mated with PHS-2 +/- males. B[a]P caused a characteristic syndrome of fetal teratological anomalies (10) , including club foot, forelimb flexure, low-set ears, kinky tail, and gastroschisis, none of which were observed in vehicle-treated dams (Fig. 1 ). B[a]P also increased the incidence of red nevus and short snout compared with vehicle-treated controls by 3.2-fold (P<0.00002) and 5.8-fold (P=0.084), respectively.



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  		Figure 1. Susceptibility of B6/129S7 mice to B[a]P teratogenicity: analysis by maternal genotype. The teratological syndrome, comprising a spectrum of anomalies (inset), is reported as the mean incidence per litter. Numbers in parentheses are the number of fetuses showing the teratological syndrome and postpartum lethality, and the number of implantations (resorptions plus fetuses) for resorptions. The asterisk indicates a significant difference compared with vehicle-treated controls (P<0.02).

The incidence of the teratological syndrome in B[a]P-treated dams was 3.8-fold higher than that in dams treated with vehicle alone (P<0.0001) (Fig. 1) . Conversely, resorptions (in utero embryonic death) were 4.3-fold higher in the vehicle-treated group compared with the B[a]P-treated group (P<0.0004). The incidence of postpartum lethality was similar in both groups.

Analysis by fetal genotype
The mean incidence per litter of the teratological syndrome in + /+ PHS-2-normal and in +/- and -/- PHS-2-deficient fetuses exposed to B[a]P was at least 2.7-fold higher than that for fetuses exposed to vehicle alone (P<0.05) (Fig. 2 ). Among the treated groups, the occurrence of teratological syndrome was at least 46% lower in -/- PHS-2 knockout fetuses compared with + /+ and +/- littermates (Fig. 2) . Similarly, among B[a]P-exposed groups, postpartum lethality was 59% lower in -/- PHS-2 knockout fetuses compared with their + /+ (P>0.05) and +/- (P<0.05) littermates (Fig. 2) . Among vehicle-treated controls, postpartum lethality did not differ by fetal PHS-2 genotype. Resorptions among B[a]P-exposed +/- and -/- fetuses were 74% (P<0.05) and 76% (P>0.05) lower than their respective vehicle-exposed controls (Fig. 2) . Although no resorptions with a + /+ genotype were observed with vehicle exposure, the small number (9) precluded an accurate interpretation in this case. Fetal body weights (vehicle, 1.07 ± 0.08 g; B[a]P, 1.04 ± 0.17 g) (± SD) were not different among the fetuses of different genotypes in the treated groups compared with vehicle-treated controls.



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  		Figure 2. Susceptibility of B6/129S7 mice to B[a]P teratogenicity: analysis by fetal genotype. The teratological syndrome, comprising the spectrum of anomalies shown in Fig. 5 , is reported as the mean incidence per litter. The numbers in parentheses are the number of fetuses for the teratological syndrome and postpartum lethality, and the number of implantations (resorptions plus fetuses) for resorptions. The asterisk indicates a significant difference compared with vehicle-treated controls of the same genotype (P<0.05), and the cross indicates a significant difference compared with respective B[a]P-exposed +/- fetuses (P<0.05).

Effect of PHS-2 genotype on B[a]P teratogenicity
To determine the teratological relevance of B[a]P bioactivation by PHS-2 as distinct from the PHS-1 isoenzyme, B[a]P teratogenicity was evaluated in + /+ PHS-2-normal and +/- PHS-2-deficient dams, all of which were mated with +/- males.

Analysis by maternal genotype
Collectively, compared with +/- PHS-2-deficient dams, the incidences of the teratological syndrome and fetal resorptions were, respectively, 3.3-fold and 4.9-fold higher in the fetuses of + /+ PHS-2-normal dams (P<0.05) (Fig. 3 ). There was no significant difference in the incidence of postpartum lethality (+/+, 26.9%; +/-, 40%) or in mean fetal body weights (+/+, 1.06 ± 0.07 g; +/-, 1.04 ± 0.17 g) (± SD).



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  		Figure 3. Embryopathic effects of B[a]P in + /+ PHS-2-normal and +/- PHS-2-deficient knockout mice. Malformations reflect the composite of specific fetal malformations (inset) that were significantly different between maternal genotypes. The numbers in parentheses are the number of fetuses for malformations and postpartum lethality, and the number of implantations for resorptions. The asterisk indicates a difference from +/+ PHS-2 -normal dams (P<0.05).

With respect to individual birth defects, compared with + /+ PHS-2 normal dams, the +/- PHS-2-deficient dams treated with B[a]P had an 80% lower incidence of fetuses with club foot (P<0.0001), a 63% lower incidence of fetuses with short snout, and an 86% lower incidence of fetuses with kinky tail (P<0.05) (Fig. 3 , inset). Differences in other anomalies analyzed independently were not statistically significant (P>0.05).

Analysis by fetal genotype
In B[a]P-treated +/- dams, the occurrence of the teratological syndrome in the +/+ PHS-2-normal and +/- PHS-2-deficient fetuses was about two times higher than that for the -/- PHS-2-deficient knockout fetuses (P<0.05) (Fig. 4 ). Although a similar pattern was observed with + /+ dams, it is difficult to compare these data because this genotype cannot yield -/- fetuses.



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  		Figure 4. Effect of fetal PHS-2 genotype on susceptibility to the B[a]P-initiated teratological syndrome and postpartum lethality. The teratological syndrome is reported as the mean incidence per litter. The number of fetuses is given in parentheses. The asterisk indicates a significant difference compared with the respective +/- fetuses from +/- dams (P<0.05). The cross indicates a significant difference compared with the +/- fetuses of the + /+ dams (P<0.05).

The apparent protection against individual B[a]P-initiated birth defects evident in -/- knockout fetuses compared with both + /+ and +/- littermates was statistically significant only for club foot, the major malformation associated with B[a]P (9) , which was completely absent in -/- fetuses (+/+, P=0.07; +/-, P<0.01) (Fig. 5 ). Although there was a trend for decreasing B[a]P-initiated forelimb flexure, red nevus, short snout, and kinky tail, going from + /+ to +/- to -/- fetuses, this trend was not statistically significant (Fig. 5) .

In B[a]P-treated +/- dams, the incidence of postpartum lethality was 2.6-fold higher for both + /+ and +/- (P<0.05) fetuses compared with their -/- PHS-2 knockout littermates (P<0.05) (Fig. 4) . As for the teratological syndrome, the postpartum lethality data for the + /+ dams (which cannot yield -/- fetuses) cannot be directly compared, but in this case the data do not completely mirror the pattern in +/- dams.

When the residual tissue from B[a]P-exposed resorbed fetuses was dissected from the uterus and the genotype determined using PCR, there appeared to be a higher incidence of + /+ and +/- resorptions in + /+ dams (resorptions: +/+, 20%; +/-, 24%) than in +/- dams (resorptions: +/+, 0%; +/-, 7%) when standardized by the number of implantations. Accordingly, the data from the two maternal genotypes could not be combined. This difference by maternal genotype was significant only for the +/- resorptions, the numbers of which were 3.5-fold higher in + /+ dams compared with +/- dams (P=0.032). However, this difference was not evident if the +/- resorptions were standardized by the total number of resorptions, rather than implantations, for the respective maternal genotypes (+/- resorptions in +/+ dams, 88%; in +/- dams, 67%) (P=0.50). In the absence of combining maternal data, the number of resorptions for each of the three possible embryonic genotypes in the +/- dams was too small for statistical comparison. The mean fetal body weight of the surviving fetuses did not differ among embryonic genotypes (+/+, 1.05 ± 0.14 g; +/-, 1.04 ± 0.15 g; -/-, 1.03 ± 0.22 g) (±SD).

Immunoblot analysis of embryonic PHS-2
PHS-2 protein was detected in embryos of GD 10–13 (Fig. 6 ). The expression of this protein in embryos during organogenesis was substantially higher than that in adult brain, the adult tissue in which this protein is known to be expressed constitutively (21) . PHS-2 was also detected at low levels in adult kidney but not in the heart (Fig. 6B ).



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  		Figure 6. PHS-2 (shown as Cox-2 in the figure) protein expression in untreated CD-1 mouse embryos during organogenesis. A) Immunoblot for embryos of gestational day (GD) 10. B) Immunoblot for embryos of GD 11–13.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
PHSs have been implicated in the bioactivation of xenobiotics to a free radical intermediate that initiates the formation of teratogenic reactive oxygen species (ROS) (22 , 23) . However, there is no direct information available on the expression of embryonic PHS isozymes during organogenesis, nor on the relative contribution of the embryonic PHS-2 isozyme, which is nonconstitutive in most adult tissues, to xenobiotic bioactivation and teratogenicity. Our goals in this study were to determine the embryonic expression of PHS-2, and its role, as distinct from PHS-1 and the cytochromes P450, in the bioactivation of B[a]P to a teratogenic reactive intermediate.

Strain is responsive to B[a]P teratogenicity
Although the C57BL/6 (B6) strain of mice is sensitive to B[a]P teratogenicity (9 , 10) , the strain of mice used in the present study has a hybrid B6/129S7 background. Accordingly, we first characterized the susceptibility of this hybrid strain to B[a]P teratogenicity. The B6/129S7 strain proved to be susceptible to the full spectrum of structural fetal birth defects, collectively termed a teratological syndrome, characteristic of B[a]P. Thus, B[a]P caused club foot, forelimb flexure, low-set ears, kinky tail, and gastroschisis, none of which were observed in vehicle-treated controls, as well as increasing the incidence of red nevus and short snout. The occurrence and significance of a characteristic pattern or syndrome of anomalies are common in chemical teratogenesis and often more revealing than are individual birth defects, as exemplified in animals and humans with fetal alcohol syndrome (24) and fetal hydantoin syndrome (25 , 26) . Although significant increases in occurrence of both the syndrome and individual anomalies provided evidence for PHS-2-catalyzed bioactivation of B[a]P when analyzed by maternal genotype, the syndrome was more revealing in the subsequent analysis by fetal genotype when subgroup numbers were smaller.

In contrast to teratological anomalies, the numbers of fetal resorptions (in utero death) analyzed by maternal genotype were higher in the vehicle-treated group compared with dams treated with B[a]P. The apparent protection against fetal resorptions in the B[a]P-treated group may be due to PHS-2 induction by B[a]P in these dams. B[a]P has been shown to induce PHS-2 mRNA and protein in oral epithelial cells in a concentration-dependent manner (27) , as was demonstrated for another polycyclic aromatic hydrocarbon, dioxin, in rat skin fibroblasts (28) . PHS-2 is necessary for implantation, as evidenced by the infertility of -/- PHS-2 knockout dams (2) . Lim and co-workers have shown that disruption of PHS-2 in mice produces multiple failures in female reproductive processes, including ovulation, fertilization, implantation, and decidualization (29) . In addition, PHS-2-derived prostacyclin activates the nuclear hormone receptor PPAR{delta} and is essential for implantation and decidualization (30) . Although the studies by Lim and co-workers (29) did not include +/- PHS-2-deficient mice, lack of even one copy of the gene may be detrimental to normal development. Thus, low levels of maternal and/or embryonic PHS-2 may increase the risk of fetal resorption, which could be alleviated by B[a]P-mediated PHS-2 induction.

PHS-2-catalyzed bioactivation contributes to B[a]P teratogenicity
To determine whether PHS-2-catalyzed bioactivation of B[a]P contributes to its teratogenicity, +/+ PHS-2-normal and +/- PHS-2 knockout females were mated with +/- males and treated with B[a]P during organogenesis.

Analysis by maternal genotype showed that, compared with +/- PHS-2-deficient dams, the incidences of the B[a]P-initiated teratological syndrome and fetal resorptions were both several times higher in + /+ PHS-2-normal dams (P<0.05) (Fig. 3) . With respect to individual birth defects, + /+ PHS-2-normal dams produced particularly higher incidences of club foot, short snout, and kinky tail. These results are consistent with higher PHS-2-catalyzed bioactivation of B[a]P to an embryopathic reactive intermediate in fetuses from + /+ PHS-2-normal dams.

A similar pattern supporting a role for PHS-2-catalyzed bioactivation of B[a]P was observed when the data were analyzed by fetal genotype, which corroborated the increased number of fetal malformations according to maternal genotype. These data indicate that even one copy of the PHS-2 gene is sufficient to support teratologically relevant B[a]P bioactivation, suggesting that high levels of PHS-2 expression will increase teratological risk. Although there was a trend for decreasing B[a]P-initiated forelimb flexure, red nevus, short snout, and kinky tail, going from + /+ to +/- to -/- fetuses, this trend was not statistically significant. Given that a gene dosage effect has been reported for PHS message and protein synthesis, a correlation between the decrease in PHS-2 alleles and embryopathies might be expected. PHS-2 mRNA and protein expressed by heterozygous PHS-2 mice are 50% of levels in congenic, wild-type mice, whereas PHS-2 knockout mice express neither PHS-2 mRNA nor PHS-2 protein (2 , 31) . Our observation that +/- fetuses are not protected compared with their + /+ littermates suggests that even a 50% level of expression of PHS-2 protein is adequate to provide teratologically relevant bioactivation of B[a]P. This pattern is reproducible, in that a similar level of embryopathic outcomes was observed for + /+ and +/- littermates in the first study showing strain susceptibility to B[a]P (Fig. 2) . The lack of statistical significance for the apparent decreasing trend for malformations among +/+, +/-, and -/- fetuses may reflect either insufficient numbers or a threshold requirement for PHS-2 in teratogen bioactivation. In either case, it appears that at least one if not both PHS-2 alleles must be lost to sufficiently reduce PHS-2-catalyzed bioactivation for measurable protection against B[a]P-initiated teratogenicity.

A similar pattern of fetal PHS-2-dependent risk was evident for postpartum lethality, the incidence of which was several times higher for both + /+ and +/- fetuses compared with their -/- PHS-2-deficient littermates. There was no difference in the incidence of postpartum lethality between + /+ PHS-2-normal and +/- PHS-2 -deficient fetuses, indicating again that only one embryonic PHS-2 allele is necessary for embryopathically relevant B[a]P bioactivation.

When the residual tissue from resorbed fetuses was dissected from the uterus and the genotype determined using PCR, the apparent differences by maternal genotype for the same embryonic genotype precluded combining data from the + /+ and +/- dams. The 3.5-fold higher incidence of +/- resorptions from +/+ dams than from +/- dams is consistent with the analysis by maternal genotype showing an increased resorption incidence in + /+ dams and further suggests an influence of maternal genotype on embryonic susceptibility. However, this difference was not evident if the data for resorption genotype was standardized by the total number of resorptions rather than implantations. A definitive resolution of this issue will require a larger study focusing on +/- dams in which a larger number of resorptions and implantations is achieved for each of the three possible embryonic genotypes.

PHS-2 is constitutive in embryos during organogenesis
Unlike the PHS-1 isoenzyme, PHS-2 is nonconstitutive but is inducible in most adult tissues. The only adult tissues that are known to express PHS-2 protein constitutively are brain (7) , vas deferens (6) , and macula densa of the kidneys (5) . Interestingly, brain and kidney were among several tissues with the lowest level of PHS-2 mRNA as measured by reverse transcriptase-PCR (RT-PCR) (32) . Recently, semiquantitative RT-PCR analysis for PHS-2 mRNA expression during fetal bladder development revealed the highest level at GD 11.5, with a progressive decline through gestation (33) . Levels at birth were similar to those seen in adult bladder. Although PHS-1 protein has been found constitutively in the embryo and uterus of the mouse from ovulation through implantation (34) , as well as on GD 9.5 (18) , there is no evidence for PHS-2 protein expression during the period of organogenesis. In the present study, PHS-2 protein was detected in embryos at GD 10–13, and the protein level did not decrease with gestational age. The expression of this protein in embryos during organogenesis was substantially higher than that in adult brain, one of the few adult tissues in which this protein is known to be expressed constitutively (7) . PHS-2 was also detected at low levels in the adult kidney, consistent with previous observations (5) , but not in the heart. The mouse PHS-2 protein was detected at a lower molecular mass than that of the ovine PHS-2 (72 kDa), which was used as the positive standard in this study. This is in agreement with the previously reported lower molecular mass for the mouse enzyme compared with the standard ovine PHS-2 (35) , which may be due to naturally occurring deglycosylation of this enzyme. Unpurified PHS-2 from tissues or cell lysate has been reported to sometimes appear between 65 and 74 kDa, also likely due to naturally occurring deglycosylation (36) . The specificity of the antibody for the PHS-2 isozyme is also consistent with its failure to react with PHS-1 (data not shown), the absence of background bands, the presence of the same molecular weight band from maternal tissues known to constitutively express PHS-2 (brain, kidney), and the absence of this band in maternal heart, which does not constitutively express PHS-2. These results provide the first direct evidence for constitutive expression of PHS-2 in mouse embryos during the critical period of organogenesis, during which embryos are susceptible to teratogenesis.

In both human and rodent adults, the carcinogenicity of B[a]P is thought to depend on, at least in part, its bioactivation by P450s, and particularly by CYP1A1 isozyme, to a toxic electrophilic reactive intermediate that covalently binds to DNA and protein (8) . However, in rodents, CYP1A1 is not constitutively expressed in any prenatal conceptual tissues or any stage of gestation investigated thus far (37) , although it is transplacentally inducible in a variety of prenatal rat and mouse tissues by methylcholanthrene/dioxin-type inducing agents (38) . Very low constitutive levels of CYP1A1 protein are reported in first- trimester human fetal liver (37) . CYP1B1, another developmentally regulated P450 (39) , is also known to metabolically activate B[a]P (40) , and low levels of CYP1B1 mRNA have been detected in late- gestation rodent fetuses (39) . Although CYP1B1 mRNA has been detected in many human fetal tissues during the period of organogenesis (37) , the protein for this enzyme has not been detected by Western blot analysis, which may mean that the message is expressed at levels too low to be of biological or toxicological importance (37) . It remains to be determined what minimal level of P450 bioactivating activity is necessary to be teratologically relevant, and this threshold may vary both for the P450 isoenzyme and the proteratogen.

In contrast to most P450s, the content and activity of at least the PHS-1 isozyme are high in rodent and human embryos during organogenesis (23) . PHS has been shown in mice and rabbits to bioactivate a number of proteratogens, including B[a]P, phenytoin and related anticonvulsants, and thalidomide, to a free radical intermediate that initiates the formation of ROS (19 , 41 42 43 44) . Direct bioactivation by PHS has been shown for most of these proteratogens (19) . The case for B[a]P is less clear, because the only pathway studied is its PHS-catalyzed bioactivation to the electrophilic 7,8-diol-9,10-epoxide reactive intermediate, or its covalent binding to DNA, using the B[a]P 7,8-diol metabolite as the substrate (1 , 13) , rather than its bioactivation to a free radical intermediate. Together with the evidence discussed above for low embryonic P450 activity in rodents, the observation from embryo culture that embryonic bioactivation of B[a]P can result in teratologically relevant ROS formation and oxidation of DNA (18) suggests that a P450-catalyzed reaction may not be a necessary prerequisite and that an epoxide may not be the teratologically relevant reactive intermediate of B[a]P. ROS initiated by the above teratogens oxidatively damage embryonic cellular macromolecules such as DNA, protein, and lipid, and this oxidative damage is thought to play an important role in teratological initiation. Nonspecific inhibitors of PHS block the embryopathic effects of these teratogens in vivo and in embryo culture, as do the free radical trapping agent phenylbutylnitrone, the antioxidants vitamin E and caffeic acid, and the antioxidative enzymes superoxide dismutase and catalase. Conversely, depletors of glutathione (GSH) and reductions in the activities of the antioxidative enzymes GSH reductase, GSH peroxidase, and glucose-6-phosphate dehydrogenase enhance teratogenicity. The teratogenicity of B[a]P and phenytoin is also enhanced in p53 knockout mice with deficient DNA repair, which implicates DNA as a teratologically important target for oxidative damage.

The present study provides the first direct evidence that the PHS-2 isozyme is strongly expressed in embryos throughout organogenesis, the critical period for susceptibility to teratogens. Because PHS-2 functions primarily in the nuclear envelope (45) , this is a particularly attractive isozyme for the proximate bioactivation of proteratogens to reactive intermediates that damage nuclear components such as DNA and proteins. The increased occurrence of B[a]P-initiated embyropathies observed in both + /+ and +/- mice compared with -/- PHS-2 knockouts indicates that, even with one gene copy, PHS-2-catalyzed bioactivation contributes substantially to the teratogenicity of ROS-initiating teratogens such as B[a]P.


   ACKNOWLEDGMENTS
 
We thank Mr. Daniel Kim for assistance in the teratological studies and the Jackson Laboratory for providing us with the sequences for the primers that were used in the PHS-2 genotyping assay.


   FOOTNOTES
 
1 Preliminary reports of this research were presented at the annual meetings of the Society of Toxicology (USA) [Toxicol. Sci. 42(1-S):121(No. 597), 1998], and the Society of Toxicology of Canada [Proc.: Student Award Poster No. 8, 1999]. These studies were supported by the Canadian Institutes of Health Research.

Received for publication April 10, 2001. Revision received February 28, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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