Introduction

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Members of the CYP4A subfamily of cytochrome P450 (P450¹) enzymes catalyze the - and -1-hydroxylation of fatty acids, including arachidonic acid, as well as arachidonic acid epoxidations (Nguyen et al., 1999). Expression of CYP4As in rodent livers and kidneys is induced by peroxisome proliferators (Sharma et al., 1989) and by physiological states such as diabetes or starvation in which fatty acid levels are elevated (Imaoka et al., 1990; Shimojo et al., 1993; Kroetz et al., 1998). Fatty acids and peroxisome proliferators bind to and activate peroxisome proliferator-activated receptor (PPAR), a member of the steroid hormone receptor superfamily (Wahli et al., 1999). PPAR binds as a heterodimer with the retinoid X receptor to enhancer elements on responsive genes, including the CYP4As (Johnson et al., 1996), resulting in their transcriptional activation. The in vivo induction of CYP4A mRNAs by chemicals, starvation, or diabetes requires PPAR, since mice lacking this receptor fail to respond to the respective stimuli (Lee et al., 1995; Kroetz et al., 1998).

CYP4A mRNAs are induced in the livers and kidneys of rats during inflammation and infection (Sewer et al., 1997), unlike most other hepatic P450 gene products, which are down-regulated (Morgan, 1997; Sewer et al., 1997). CYP4A mRNAs are also induced in the kidneys of female mice by treatment with bacterial lipopolysaccharide (LPS) treatment, and this effect is absent in PPAR-null mice, indicating that the renal induction is PPAR-dependent (Barclay et al., 1999). However, CYP4A mRNAs are down-regulated in female mouse liver after LPS treatment (Barclay et al., 1999), and therefore the participation of PPAR in hepatic CYP4A induction by inflammation in the rat remains to be elucidated. To gain further insight into this question, in the present study we examined the sex dependence of induction of CYP4As by inflammatory stimuli in the rat. A characteristic feature of hepatic CYP4A induction by peroxisome proliferators in this species is a pronounced sex difference (females are refractory) (Sundseth and Waxman, 1992).

Our previous work showing induction of renal and hepatic CYP4A mRNAs by inflammatory stimuli was conducted in Fischer 344 (F344) rats (Sewer et al., 1997). However, we also reported that hepatic induction of CYP4A2 mRNA by LPS treatment was absent in Sprague-Dawley (S-D) rats and that the hepatic induction of CYP4A1 and CYP4A3 mRNAs in this strain was attenuated relative to the effect seen in F344 rats (Sewer et al., 1997). Because a significant strain difference could be a potential tool to address the mechanism of CYP4A induction, a second goal of this study was to solidify this preliminary observation and to determine whether such a strain difference was restricted to LPS as the inflammatory stimulus.

As noted above, CYP4As are induced by starvation in a PPAR-dependent manner. LPS causes a reduction in food intake in mice (Kozak et al., 1994), but this hypophagia is not responsible for the induction of renal CYP4A by LPS because mice pair-fed with LPS-treated animals did not demonstrate renal CYP4A induction (Barclay et al., 1999). However, this observation did not rule out the possibility that hypophagia could contribute to induction of hepatic CYP4As in the rat, and the role of hypophagia in the response to particulate irritants has not been investigated. The present study addresses these questions as well. Our results are consistent with a sex-specific, PPAR-mediated induction of CYP4A mRNAs in rat liver, but the previously reported strain difference was not detected. We found that the induction of CYP4A expression by LPS in the rat is not caused by hypophagia, whereas the effect of the particulate irritant BaSO₄ appears to be mainly via an effect on the animals' feeding behavior.

	Materials and Methods

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Animals and Treatments. All procedures were approved by the Institutional Animal Care and Use Committee of Emory University. Male or female S-D or F344 rats (Harlan Inc., Indianapolis, IN) were allowed to acclimatize to the Animal Care Facility for at least one week after delivery, before treatment. They were 8 to 10 weeks old at the time of injection. Rats received a single i.p. injection of either 1 mg/kg (0.15 mg/ml) chromatographically pure Escherichia coli LPS, serotype 0127:B8 (Sigma, St. Louis, MO), dissolved in sterile 0.9% saline; 3 g/kg (0.45 mg/ml) BaSO₄ suspended in saline; or an equivalent volume of vehicle. We have shown that this dose of LPS produces a maximal suppression of total P450 and CYP2C11 content in rat liver (Morgan 1989) and induces CYP4A expression in rat liver and kidney (Sewer et al., 1996, 1997). The dose of BaSO₄ used was optimized previously for maximal CYP4A induction (Sewer et al., 1997). Animals were sacrificed by CO₂ asphyxiation 24 h after injection. Tissues were harvested and used immediately or flash-frozen in liquid nitrogen and stored at 80°C.

RNA Preparation and Northern Blotting. Total RNA was prepared from fresh or frozen liver or kidney by acid-phenol extraction (Chomczynski and Sacchi, 1987). Samples were stored at 80°C until analysis. RNA concentrations were determined by absorbance at 260 nm. For Northern blot assays, RNA was fractionated on a 1% agarose gel electrophoresis in the presence of 5% formaldehyde and transferred to nylon transfer membrane filters (Sambrook et al., 1989) (MagnaGraph, Micron Separations, Inc., Westboro, MA). Blots were prehybridized, hybridized, and washed as described below. The amount of probe bound to the filter was quantitated using a Molecular Dynamics (Sunnyvale, CA) 445si phosphorimager and ImageQuant software. Results were normalized to the content of glyceraldehyde-3-phosphate dehydrogenase (GAP) mRNA or rRNA using the probes described below.

Probes and Hybridization Conditions. The mRNAs for CYP4A1, -4A2, and -4A3 were detected using custom synthesized oligonucleotide probes (Life Technologies Inc., Rockville, MD) designed to recognize regions of the 3'-untranslated regions of their mRNAs that have low sequence similarity. Thus, the CYP4A1 and CYP4A3 probes (Fig. 1) are complementary to a region of their mRNAs that is expressed as part of exon 12 but which is recognized as an intron, spliced, and removed in CYP4A2 (Helvig et al., 1998). The CYP4A2 probe spans this intron and therefore is specific for this mRNA (Fig. 1). The sequences of the probes were as follows: CYP4A1, 5'-GGTATGGGAAGGGTGCTGGCTTTAAAGCAGAGGAATATTC-3'; CYP4A2, 5'-ACACACACAAGCTGGGAAGGTGTCTGGAGTAAAAGCTTTGG-3'; and CYP4A3, 5'-AATTAGCCAGTAACAAATGCAGGTATTGCAGGCAGCAGAC-3'. CYP4A1 mRNA was also detected using the 3' SacI-EcoRI fragment of its cDNA (GenBank accession no. X07259; Earnshaw et al., 1988). Fibrinogen-chain and GAP mRNAs were detected using their respective cDNAs. The fibrinogen-cDNA was kindly donated by Dr. Gerald Fuller (University of Alabama at Birmingham, Birmingham, AL); the GAP cDNA was purchased from the American Type Culture Collection (Manassas, VA). Rat 28S rRNA was detected using an oligonucleotide probe (Barbu and Dautry, 1989).

View larger version (54K): [in this window] [in a new window]   Fig. 1.   Portion of the 3'-untranslated region of rat CYP4A mRNAs. The underlined nucleotides correspond to the portions of the sequence targeted by the oligonucleotide probes used in this study.

Data Presentation and Statistical Analysis. The Northern blot assay is semiquantitative only. Therefore, no attempt has been made to express measurements in any absolute units. Values for each experiment were calculated as a percentage of the normalized mean value (arbitrary units) for an appropriate control group. All results are expressed as the mean ± S.E.M. for each group. The numbers of animals used in each experiment are given in the figure legends. The number of observations in some groups was lower than the number of animals due to loss of samples during preparation and/or the assay procedure. Statistical analyses were performed using the Quick Statistica package (StatSoft, Inc., Tulsa, OK). One-way analysis of variance and the Student Newman-Keuls test were used to determine differences among treatment groups. In cases where the variances of the experimental groups were not equivalent, the Mann-Whitney U test was used instead. The null hypothesis was rejected at P < 0.05.

	Results

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Specificity of the CYP4A Probes. Figure 2 shows the specificities of the probes used to detect the CYP4A mRNAs, as judged by the known tissue and sex-specific expression of these genes. Thus, all three CYP4A mRNAs are induced by clofibrate. Both CYP4A2 and CYP4A3 are expressed at higher levels in kidney than in liver. CYP4A3 shows no sex specificity, whereas CYP4A2 is male-specific. The CYP4A1 oligonucleotide probe detected only low levels of constitutive expression of this mRNA in both liver and kidney, independent of gender (Fig. 2). Identical results were obtained using the CYP4A1 cDNA probe (Fig. 2). The CYP4A1 cDNA probe was selected for use in subsequent experiments because it was found to give a higher signal to noise ratio in most blots.

View larger version (74K): [in this window] [in a new window]   Fig. 2.   Northern blots showing specificities of the CYP4A probes. Northern blots of sexually mature male or female F344 rat liver or kidney RNA (10 µg per sample) were hybridized with the indicated probes. Each sample was pooled from the tissues of two animals. M, male; F, female; Clo, male rats injected with a single dose of clofibrate (400 mg/kg) 24 h before sacrifice. Each blot was probed with the indicated CYP4A probe and subsequently stripped and reprobed with the GAP cDNA.

Role of Hypophagia in LPS and BaSO₄ Induction of CYP4A mRNAs. To determine the role, if any, of hypophagia in the induction of hepatic and renal CYP4A mRNAs by LPS, the food intake of saline- and LPS-treated rats was measured at 6-h intervals. The next day, animals in the pair-fed group were given food equal to the amount eaten by the LPS-treated rats during the equivalent time intervals. It was necessary to divide the treatment period into 6-h intervals to approximate the eating pattern of the LPS-treated rats. Otherwise, the pair-fed rats could eat their entire ration in a short period at the beginning of the experiment.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of LPS, pair feeding, and starvation on hepatic CYP4A mRNA expression. Groups of four to five, 8-week-old (198 ± 8 g b.wt.), male F344 rats were treated as described under Materials and Methods and sacrificed at the indicated times. CYP4A mRNA expression was measured by Northern blotting and normalized to the rRNA contents of the samples. Values are the means of four to five observations per group. The data for one animal in the 24-h LPS group was discarded because it deviated by more than 2 standard deviations from the group mean. *Significantly different from control group mean; significantly different from pair-fed group mean.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Effect of LPS, pair feeding, and starvation on renal CYP4A mRNA expression. Groups of four to five male F344 rats were treated as described under Materials and Methods and sacrificed at the indicated times. CYP4A mRNA expression was measured by Northern blotting and normalized to the rRNA contents of the samples. Values are the means of four to five observations per group. *Significantly different from control group mean; significantly different from pair-fed group mean.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   Effect of BaSO4 and pair feeding on hepatic and renal CYP4A mRNA expression. Groups of six male S-D rats (8 weeks old, 284 ± 13 g b.wt.) were treated as described under Materials and Methods and sacrificed at the indicated times. CYP4A mRNA expression was measured by Northern blotting and normalized to the GAP mRNA contents of the samples. Values are the means of four to six observations per group. Fib, fibrinogen. *Significantly different from control group mean; significantly different from pair-fed group mean.

Sex- and Strain-Dependence of CYP4A mRNA Induction by LPS and BaSO₄. The effects of LPS or BaSO₄ treatment on hepatic and renal CYP4A expression were compared in male and female rats of the F344 and S-D strains. Figure 6 shows Northern blots from the livers and kidneys of F344 rats. Figures 7 and 8 contain the quantitative data from this strain. Because the responses of CYP4A mRNAs in livers and kidneys of S-D rats of either sex were very similar to those of F344 rats, only the results for F344 rats are shown. LPS and BaSO₄ significantly induced all three CYP4A mRNAs in the livers of F344 male rats (Fig. 7). The effects of LPS or BaSO₄ treatment in male S-D rat livers were similar to those in the F344 animals, except that LPS treatment of male S-D rats produced only a trend toward an increase in CYP4A2 mRNA (LPS, 239 ± 32; control, 100 ± 74; P < 0.08) (not shown). LPS treatment failed to induce the expression of any CYP4A mRNA in the livers of female rats of either strain (F344, Fig. 7; S-D, results not shown). BaSO₄ treatment produced elevations in CYP4A1 and CYP4A3 expression in female F344 and S-D rats, but these were smaller than the effects seen in the corresponding males (F344, Fig. 7; S-D, results not shown).

View larger version (69K): [in this window] [in a new window]   Fig. 6.   Sex dependence of CYP4A mRNA induction by LPS and BaSO4 in F344 rat liver and kidney. Groups of five male (241 ± 10 g) and female (158 ± 11 g) F344 rats, 9 to 10 weeks old, were treated with LPS or BaSO4 as described under Materials and Methods. Twenty-four hours after treatment, total RNA was prepared and analyzed by Northern blotting. The samples shown are from individual rat livers.

View larger version (31K): [in this window] [in a new window]   Fig. 7.   Sex dependence of CYP4A mRNA induction by LPS and BaSO4 in F344 rat liver. Male and female F344 rats were treated with LPS or BaSO4 as described under Materials and Methods. Numbers, ages, and weights of F344 rats are given in Fig. 6. Twenty-four hours after treatment, total RNA was prepared, and the expression of CYP4A and fibrinogen mRNAs was analyzed by Northern blotting. Values are the means of four to five observations per group and are normalized to the GAP mRNA contents of the samples. *Significantly different from control group mean.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Sex dependence of CYP4A mRNA induction by LPS and BaSO4 in F344 rat kidney. Expression of CYP4A mRNAs was measured 24 h after treatment with LPS or BaSO4. The animals and treatments were the same as described in Fig. 7. Values were normalized to the GAP mRNA contents of the samples. *Significantly different from control group mean.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Our previous pair-feeding study in the mouse showed that the induction of renal CYP4A caused by LPS treatment was not caused by LPS-evoked hypophagia (Barclay et al., 1999). The results presented here indicate that this is also true of both hepatic and renal CYP4A induction in the rat. Pair feeding did not significantly elevate hepatic CYP4A1 and CYP4A2 or renal CYP4A1 and CYP4A3 expression. Although increases in hepatic CYP4A3 (Fig. 3) and renal CYP4A2 were observed with pair feeding at the 24-h time point, in both cases it can be seen that LPS treatment induced these mRNAs at the earlier (12-h) time when there was no effect of pair feeding. Thus, although LPS-induced hypophagia may produce some effect on CYP4A expression, we conclude that it is not the major mechanism. We have speculated previously that induction of CYP4As following LPS treatment is caused by generation of prostaglandins or leukotrienes during the inflammatory response, leading to activation of PPAR (Barclay et al., 1999).

Treatment of rats with the particulate irritant BaSO₄ produces a more profound effect on food intake over the first 24 h than does LPS treatment (21% of control for BaSO₄, 46% for LPS; see Tables 1 and 2). BaSO₄ treatment, or pair feeding with the BaSO₄-treated animals, had similar effects on CYP4A expression in rat liver and kidney (Fig. 5), indicating that the induction of hepatic and renal CYP4As caused by BaSO₄ treatment is mainly due to irritant-induced hypophagia. Thus, the threshold for reduced food intake causing a significant induction of CYP4A expression lies between 21% (Table 2, Fig. 5) and 46% (Table 1, Figs. 3 and 4).

Interestingly, although control rats ate only 25 to 29% of their daily intake in the first 12 h (and only 1-3% in the first 6 h, Tables 1 and 2), food deprivation during this short period caused significant induction of hepatic and renal CYP4As (starved animals, 12-h time point, Figs. 3 and 4). Whereas we observed no further induction over the next 12 h, Kroetz et al. (1998) reported that the induction of hepatic CYP4As at 48 h of starvation was more than double that observed at 24 h. Taken together, these observations suggest that the time course of CYP4A induction caused by starvation is biphasic, with peaks at 12 and 48 h.

Induction of hepatic CYP4A mRNAs by either LPS or BaSO₄ treatment showed a clear sex dependence in rats of both strains tested (F344, Fig. 7; S-D, results not shown). Induction was either absent, or the responses were significantly smaller, in female rats. Sundseth and Waxman (1992) reported that the peroxisome proliferator clofibrate (400 mg/kg daily for 3 days) failed to induce CYP4A3 and CYP4A2 in female rats and that CYP4A1 was induced to a lesser extent in females than in males. Females were also less responsive to the induction of peroxisome marker enzymes than were males (Sundseth and Waxman, 1992). Thus, the hepatic CYP4A inducers LPS, BaSO₄ (hypophagia), and peroxisome proliferators share the common characteristic of being male-specific or male-dominant, indicating that the mechanism of induction by each of these agents contains a common sex-dependent component. Since hepatic induction of CYP4As by peroxisome proliferators requires PPAR (Lee et al., 1995), our results are consistent with a role of PPAR in the hepatic induction of CYP4A by LPS in the rat, as well.

In contrast to the liver, we did not observe any clear sex dependence of renal CYP4A induction in the kidney. In S-D rats, we found that LPS or BaSO₄ treatment induced only CYP4A1 mRNA, and this occurred to the same extent in either sex (not shown). In F344 rats, this effect was male-specific (Fig. 8). In contrast, CYP4A3 expression was induced by LPS treatment in F344 females only. Clearly, induction of renal CYP4A mRNAs by inflammatory stimuli is of smaller magnitude than that observed in liver (Fig. 5; compare Figs. 3 and 4, 7 and 8), a characteristic that is shared with peroxisome proliferators (Sharma et al., 1989). The small magnitude of the renal effects undoubtedly contributes to the variability observed in the responses of individual CYP4As in different experiments (e.g., renal CYP4A2 and 4A3 were significantly induced by LPS in male F344 kidneys in the experiment in Fig. 4; these effects did not reach significance in the experiment in Fig. 8). More experiments with much larger numbers of animals would therefore be needed to determine whether sex and strain differences in renal CYP4A induction exist. The observed induction of CYP4A1 and CYP4A3 mRNAs in female rat kidneys (F344, Fig. 8; S-D, results not shown) is consistent with our previous report of a strong PPAR-dependent induction of renal CYP4A10 and CYP4A14 mRNAs in female mice (Barclay et al., 1999). To our knowledge, the sex dependence, if any, of renal CYP4A induction by peroxisomal proliferators in rats has not been reported, although the induction of renal peroxisomal enzymes acyl-CoA oxidase and bifunctional enzyme by dehydroepiandrosterone showed no marked sex difference (Yamada et al., 1991).

In a previous publication, we referred to unpublished data indicating that hepatic induction of CYP4A mRNAs in male S-D rats by LPS treatment was absent (CYP4A2) or reduced (CYP4A1, -4A3) compared with that seen in F344 rats (Sewer et al., 1997). The present results provide no evidence for this strain difference suggested by the previous preliminary study. The effects of LPS and BaSO₄ on hepatic CYP4A mRNAs were similar in both strains, except that the induction of CYP4A2 mRNA by LPS in S-D rats just failed to achieve statistical significance. This lack of a strain difference is perhaps not surprising, given that no major strain difference has been reported in the effects of peroxisome proliferators in rats and that the available evidence (present study; Barclay et al., 1999) suggests that induction of hepatic CYP4As following LPS treatment is PPAR-dependent.

In our characterization of the oligonucleotide and cDNA probes used to measure the CYP4A mRNAs (Fig. 2), the observed sex and tissue specificities of CYP4A2 and -4A3 expression detected by our probes were in general agreement with those of other groups (Sundseth and Waxman, 1992; Helvig et al., 1998). In contrast, Helvig et al. (1998) found high levels of expression of CYP4A1 in female S-D rat kidney using a SacI-EcoRI cDNA probe, whereas we observed very low basal expression of CYP4A1 mRNA in kidneys of F344 rats of either sex. The disparity was not caused by a strain or age difference: we repeated this experiment using RNA from 8- and 13-week-old S-D rats and found essentially the same results (not shown) seen in Fig. 2 using tissues from F344 rats. We obtained the same results using either an end-labeled oligonucleotide probe or a random primer-labeled cDNA fragment, both corresponding to a portion of the 3'-untranslated region of the CYP4A1 mRNA. Helvig et al. (1998) used the same cDNA fragment, labeled by nick translation. Therefore, the difference in our findings is unexplained. However, our results agree with those of Sundseth and Waxman (1992), who used an oligonucleotide probe.

In conclusion, we have shown that the induction of hepatic CYP4A after treatment of rats with LPS or the particulate irritant BaSO₄ is male-specific, consistent with participation of PPAR in these responses. We have also demonstrated that whereas the induction of CYP4A expression caused by BaSO₄ treatment is mainly due to hypophagia, the induction by LPS treatment is not caused by the reduction in food intake in these animals.