Androgen-Dependent Differences in the Amounts of CYP mRNAs in the Pig Kidney

Abstract

We previously reported androgen-dependent sex and breed differences in the amounts of mRNAs of CYP isoforms in the pig liver. To clarify whether there are such sex and breed differences in the kidney, we examined the amounts of several CYP mRNAs in the kidney using both sexes of 5-month-old Landrace, Meishan and/or their crossbred F₁ (LM and ML) pigs. Significant sex differences in the amounts of several CYP mRNAs were found: male < female for CYP2A19 and CYP3A29; and male > female for CYP4A24/25 in all the breeds. Sex differences in the amount of CYP2B22 mRNA (male < female) and in CYP2C33 and CYP2C49 mRNAs (male > female) were also observed in all the breeds except Landrace pigs. Furthermore, a significant sex difference (male < female) in CYP3A46 mRNA was only found in LM and ML pigs. No significant sex differences were found in either Landrace or Meishan pigs for CYP1A1, CYP1A2 and CYP4B1 mRNAs. The amounts of CYP2C33 and CYP4A24/25 mRNAs in males were higher in Meishan pigs than in Landrace pigs. Additional experiments using pigs treated by castration and/or testosterone propionate indicated that sex and breed differences in the amounts of those CYP mRNAs were, at least in part, dependent on the levels of serum testosterone. Furthermore, the effects of androgen on the amounts of CYP mRNAs in the kidney did not necessarily correlate with those in the liver, suggesting that there is a tissue-selective factor responsible for the androgen-related expression of CYP genes.

INTRODUCTION

Drug metabolizing enzymes (DMEs), such as CYPs, sulfotransferases (SULTs), and uridine 5′-diphosphate (UDP)-glucuronosyltransferases (UGTs), play an important role in the metabolism of xenobiotics, including drugs and endogenous compounds such as steroid hormones and lipids.^1,2) Domestic pigs (Sus scrofa domesticus), including mini pigs, are important in pharmacological and toxicological studies because of the similarity of DME function between humans and pigs, particularly CYPs.^3–7) We have previously reported sex and breed differences in the amounts of hepatic DME mRNAs, including CYP isoforms, between Landrace and Meishan pigs, and we further demonstrated that serum androgen level is one of the critical factors producing these differences.⁸⁾

The kidney as well as the liver is the important tissue responsible for the metabolism and excretion of xenobiotics including drugs and endogenous compounds, and is one of the most frequent targets for drug-induced toxicity.^9,10) It has been reported that CYP isoforms belonging to the CYP1, CYP2, CYP3 and CYP4 families exist in the pig kidney.^11–15) Most of those CYP isoforms play an important role in the metabolism of xenobiotics and/or lipids in experimental animals and humans.^9,10,16,17) Furthermore, the expression of Cyp4a and Cyp4b isoforms in the mouse kidney is regulated by androgen.^18–21)

In the present study, we first examined sex and breed differences in the amounts of CYP mRNAs (CYP1A1, CYP1A2, CYP2A19, CYP2B22, CYP2E1, CYP2C33, CYP2C49, CYP3A22, CYP3A29, CYP3A46, CYP4A24/25 and CYP4B1) in the kidney of Meishan, Landrace, and their crossbred F₁ pigs. Furthermore, we examined whether androgen is a critical factor determining the sex and breed differences in the amounts of those CYP mRNAs in the pig kidney, by experiments with castration and/or the administration of testosterone propionate (TP).

MATERIALS AND METHODS

Animals

Landrace, Meishan and their crossbred F₁ (LM, female Landrace × male Meishan; ML, female Meishan × male Landrace) pigs were used, the same as those used in our previous studies.²²⁾ Briefly, pigs were kept at the National Institute of Livestock and Grassland Science, Tsukuba, Japan. All pigs were fed a commercial grain diet and provided with water ad libitum. Pigs were killed by electrical stunning followed by exsanguination between 10:00 and 11:00 am. Some male Landrace and Meishan pigs were castrated at the age of 1 month and euthanized at the age of 5 months. A portion of the kidney or the liver from each pig was quickly removed, frozen in liquid nitrogen, and kept at −80 °C until subsequent analyses.

This study was approved by the Animal Care Committee of the National Institute of Livestock and Grassland Science (Nos. 1711B060, 1811B032, 1911B068, 20B005NILGS). All animals were handled humanely under the guidelines of the Committee.

Androgen Treatment

As described in previous reports,^23,24) testosterone propionate (TP, Sigma Chemical Co., St. Louis, MO, U.S.A.) dissolved in corn oil was injected intramuscularly five times at 48-h intervals into the rear leg of each pig, at a dose of 10 mg/kg body weight; the pigs were euthanized 24 h after the final injection.

Amount of Serum Testosterone

The amount of serum testosterone in individual male pigs was measured using the Testosterone enzyme-linked immunosorbent assay (ELISA) Kit (Enzo Life Sciences Inc., NY, U.S.A.) according to the manufacturer’s instructions, as previously described.²⁴⁾ Additionally, the data obtained in our previous studies were also used.^24,25)

Amounts of CYP mRNAs

Total RNA was prepared from the individual tissues using TRIzol Reagent (Invitrogen Corp., Carlsbad, CA, U.S.A.), and used to determine the amounts of CYP1A1, CYP1A2, CYP2A19, CYP2B22, CYP2E1, CYP2C33, CYP2C49, CYP3A22, CYP3A29, CYP3A46, CYP4A24/25 and CYP4B1 mRNAs. The amounts of these mRNAs were assessed using real-time RT-PCR using a 7500 Real Time PCR System and SYBR green master mix (PE Applied Systems, Tokyo, Japan), as previously described,²⁵⁾ except for CYP3A mRNA. The amounts of CYP3A22, CYP3A29 and CYP3A46 mRNAs were examined using Eagle Taq Master Mix with ROX (Roche Molecular Systems, Inc., Branchburg, NJ, U.S.A.) as reported previously.²²⁾ The primer sets and TaqMan probes used in the present study were the same as those in our previous studies^22,25) except for CYP1A1, CYP1A2, CYP4A24/25 and CYP4B1, where the primer sequences were as follows: CYP1A1, 5′-ACT TCA TCC CTA TCC TCC GTT ACC-3′ (forward) and 5′-GCA TTC TCG TCC ATC CTC TTG TC-3′ (reverse); CYP1A2, 5′-GCT ATC GGG ACT TTG ACA AGA ACT-3′ (forward) and 5′-CCA GGA GAT GGC TGT GGT AAT TG-3′ (reverse); CYP4A24/25, 5′-CAC CGT GCC GCC AGG ATA-3′ (forward) and 5′-CAA GGA GGA GGA TGT CCA GGA A-3′ (reverse); CYP4B1; 5′-GAG CAC CAG CAT CGT TGT CG-3′ (forward) and 5′-TCC CCA GAT CCT CCC CCT G-3′ (reverse). Ribosomal protein L7 (RPL7) was used as an internal standard. The amount of each cDNA was assessed by the relative standard curve method according to PE Applied Biosystems User Bulletin #2 (1997). Standard curves to determine the amounts of the CYP mRNAs were generated using an RT-reaction mixture with total RNA from the liver of 5-month-old female Landrace pigs, or from the kidney of 5-month-old female Landrace or Meishan pigs, according to the purpose.

Statistical Analyses

Statistically significant differences were assessed by Tukey’s post hoc test after ANOVA or Student’s t-test. Correlations between the indicated parameters were determined by regression analysis.

RESULTS

Amounts of CYP mRNAs in the Kidney of Landrace and Meishan Pigs

The amounts of CYP mRNAs were first measured in the kidney of both sexes of 5-month-old Landrace and Meishan pigs. To compare the amount of each CYP mRNA between the kidney and the liver, the amount of each CYP mRNA in the kidney was expressed as a relative ratio to the corresponding amount in the liver of 5-month-old female Landrace pigs, and the results are shown in Table 1.

Table 1. Relative Amounts of mRNAs of Renal CYP Isoforms in 5-Month-Old Landrace and Meishan Pigs*

CYPs	Landrace pigs		Meishan pigs
	Male	Female	Male	Female
CYP1A1	0.013 ± 0.006	0.025 ± 0.018	0.006 ± 0.003	0.006 ± 0.002c)
CYP1A2	0.008 ± 0.004	0.006 ± 0.002	0.002 ± 0.001d)	0.003 ± 0.001
CYP2A19	0.042 ± 0.036b)	0.178 ± 0.045	0.0002 ± 0.0002	0.014 ± 0.006d)
CYP2B22	0.275 ± 0.146	0.314 ± 0.115	0.006 ± 0.002d)	0.018 ± 0.005d)
CYP2C33	0.513 ± 0.225	0.329 ± 0.088	0.760 ± 0.189b)	0.174 ± 0.038
CYP2C49	0.0003 ± 0.0002	0.0004 ± 0.0003	0.487 ± 0.260b),d)	0.055 ± 0.031d)
CYP3A29	0.0028 ± 0.0015	0.007 ± 0.003	0.049 ± 0.023b),d)	0.129 ± 0.042d)
CYP3A46	0.073 ± 0.054	0.072 ± 0.070	nd	nd
CYP4A24/25	7.736 ± 2.180b)	1.602 ± 0.750	17.78 ± 5.644b),d)	1.429 ± 0.266
CYP4B1	14.319 ± 6.004	10.052 ± 1.076	4.570 ± 1.570d)	3.965 ± 1.211c)

*Amount of each mRNA was expressed as relative ratio to the amount of the corresponding CYP mRNA of female Landrace pig liver. Standard curves to determine amounts of CYP mRNAs were generated using an RT-reaction mixture from livers of 5-month-old female Landrace pigs except for determination of amounts of CYP4A24/25 and CYP4B1 mRNAs. To determine amounts of those mRNAs, standard curves were generated using an RT-reaction mixture with total RNA from kidneys of 5-month-old female Landrace pigs. Each group contains 6 pigs. Significant differences were evaluated using Tukey’s post hoc test. ^{a), b)} Significant sex differences in each breed: ^a)  p < 0.05, ^b) p < 0.01. ^{c), d)} Significant breed differences between sex matched Meishan and Landrace pigs: ^c) p < 0.05, ^d) p < 0.01. nd, not detected.

In all the pigs examined, the amounts of CYP4A24/25 and CYP4B1 mRNAs in the kidney were much higher than those in the liver, while those of the other CYPs were the opposite (Table 1). In addition, although CYP2E1 and CYP3A22 mRNAs were detected in the kidney, those amounts were below one-thousandth of those in the liver in each breed (data not shown).

In both breeds of pig, the amounts of CYP2A19 and CYP3A29 mRNAs were lower in males than females (Table 1, Fig. 1). Conversely, the amount of CYP4A24/25 mRNA in both breeds of pig was significantly higher in males than in females. Furthermore, in Meishan pigs, the amounts of CYP2C33 and CYP2C49 mRNAs were significantly higher in males than in females. No such sex differences in the amounts of CYP2C33 and CYP2C49 mRNAs were observed in Landrace pigs. No significant sex differences in the amounts of CYP1A1, CYP1A2, CYP2B22 and CYP4B1 mRNAs were observed in either breed of pig, although a sex difference in the amount of CYP2B22 mRNA in Meishan pigs was significant by Student’s t-test. Furthermore, CYP3A46 mRNA was detected in both sexes of Landrace pigs, while it was below the detection limit in both sexes of Meishan pigs.

Fig. 1. Effects of Castration and/or TP Administration on Amounts of CYP mRNAs in 5-Month-Old Pig Kidney

TP was injected intramuscularly into 5-month-old castrated male and intact female Landrace and Meishan pigs, as described in the Materials and Methods. The amount of each CYP mRNA in the kidney was determined by real time RT-PCR and normalized to that of RPL7 as an internal standard. Standard curves were generated using an RT-reaction mixture with total RNA from kidneys of 5-month-old female Landrace pigs, except for the determination of the amount of CYP2C49 mRNA, where standard curves were generated using an RT-reaction mixture with total RNA from the kidneys of 5-month-old female Meishan pigs. Each column represents the mean of each experimental group, and each bar represents the standard deviation (S.D.) of the mean (n = 6 for intact male and female Landrace and Meishan pigs; n = 3 for others). ^a,b Significant differences from the corresponding intact male pigs were assessed by Tukey’s post hoc test: ^a p < 0.05, ^b p < 0.01. ^c,d Significant differences from the corresponding TP untreated pigs, as assessed by Student’s t-test: ^c p < 0.05, ^d p < 0.01. Squares in CYP2B22 and CYP3A29 indicate Meishan and Landrace pigs, respectively, with magnification.

Breed differences in the amounts of several CYP mRNAs were also found. The amounts of CYP2B22 and CYP4B1 mRNAs in Landrace pigs were significantly higher than those in sex-matched Meishan pigs, while those of CYP2C49 and CYP3A29 mRNAs were the opposite. A significant breed difference (Meishan > Landrace) in the amounts of CYP2C33 and CYP4A24/25 mRNA were also observed in males, but no such difference was found in females. Furthermore, the breed differences (Landrace > Meishan) in the amounts of CYP1A1, CYP1A2 and CYP2A19 mRNAs between sex-matched Landrace and Meishan pigs were also observed.

Effect of Androgen on the Gene Expression of Representative Sex-Related CYPs

To clarify the androgen dependency of the gene expression of CYPs (CYP2A19, CYP2B22, CYP2C33, CYP2C49 and CYP3A29) in the kidney, the effects of castration and/or TP treatment were examined using 5-month-old Meishan and Landrace pigs (Fig. 1). In castrated males of both breeds, the amount of CYP2A19 mRNA was higher than those in the corresponding intact males. Furthermore, TP treatment of castrated males and intact females of both breeds of pig resulted in a decrease in the mRNA amount. The amounts of CYP2B22 and CYP3A29 mRNAs increased in castrated Meishan pigs, but not in castrated Landrace pigs, compared with the corresponding intact males. TP treatment of castrated males and intact females of both breeds of pig resulted in a decrease in the amounts of those mRNAs, although no significant effects of TP on CYP2B22 mRNA in castrated Meishan and CYP3A29 mRNA in castrated Landrace pigs were observed because of large inter-individual difference.

The amount of CYP2C33 mRNA decreased in castrated Meishan pigs, but not in castrated Landrace pigs, compared with the corresponding intact males, and TP treatment of castrated males and intact females of both breeds of pig resulted in an increase in the mRNA amount. In Meishan pigs, the amount of CYP2C49 mRNA was also decreased by castration, and TP treatment of castrated males and intact females led to an increase in its amount. No such effects of castration or TP treatment were observed in Landrace pigs.

To date, there are no reports concerning the constitutive amounts of CYP4A24/25 and CYP4B1 mRNAs and sex differences in their amounts in the pig liver, although a clear sex difference (male > female) in the amount of CYP4A24/25 mRNA is observed here in the pig kidney (Table 1). Therefore, to clarify the effects of androgen on the expression of those mRNAs in the kidney and the liver of both Landrace and Meishan pigs, we examined not only the effects of castration but also the effects of TP treatment of castrated males and intact females (Fig. 2). The amount of CYP4A24/25 mRNA in the kidney was decreased in both Landrace and Meishan pigs by castration, and TP treatment of the castrated males and intact females of both breeds of pig resulted in an increase in its amount. However, in the liver of both breeds of pig, no such effects of castration were observed, and also no sex difference in the amount of CYP4A24/25 mRNA was observed. Likewise, no significant effect of TP-treatment on the amount of hepatic CYP4A24/25 mRNA was observed in both breeds of pig, although increase of its amount by TP treatment was observed in only castrated males of Landrace pigs. Furthermore, CYP4B1 mRNA showed no sex difference in both the kidney (Fig. 2 and Table 1) and the liver for each breed of pig. In both breeds of pig, the amount of CYP4B1 mRNA was not significantly changed by castration and/or TP treatment (Fig. 2).

Fig. 2. Effects of Castration and/or TP Administration on the Amounts of CYP4A24/25 and CYP4B1 mRNAs in the Kidney and Liver of 5-Month-Old Pigs

TP was injected intramuscularly into 5-month-old castrated male and intact female Landrace and Meishan pigs, as described in the Materials and Methods. The amount of each CYP mRNA in the kidney and the liver was determined by real time RT-PCR and normalized to that of RPL7 as an internal standard. Standard curves were generated using an RT-reaction mixture with total RNA from the kidneys of 5-month-old female Landrace pigs. Each column represents the mean in each experimental group, and each bar represents the S.D. of the mean (n = 6 for intact male and female Landrace and Meishan pigs; n = 3 for others). ^a Significant differences from the corresponding intact male pigs were assessed by Tukey’s post hoc test: ^a p < 0.01. ^b,c Significant differences from the corresponding TP untreated pigs, as assessed by Student’s t-test: ^b p < 0.05, ^c p < 0.01.

We have previously found that TP treatment resulted in an increase in the amount of CYP3A46 mRNA in the liver of Landrace pigs.²²⁾ Therefore, the effects of TP treatment and castration on the mRNA amount in the kidney were further examined using 5-month-old Landrace pigs (Fig. 1). Although castration of males had no significant effect on the amount of CYP3A46 mRNA in the kidney, TP treatment of castrated males and intact females resulted in a decrease in the amount of CYP3A46 mRNA. Furthermore, CYP3A46 mRNA was below the detection limit in both the kidney and the liver of all the Meishan pigs treated with castration and/or TP.²²⁾

Genetic Factors Responsible for Sex-Related Expression of CYP mRNAs

We have previously reported that sex-related expression of CYP mRNAs in the pig liver is, at least in part, dependent on the amount of serum testosterone,⁸⁾ and that the amount is determined by autosomal dominant inheritance.²⁵⁾ Therefore, using F₁ progeny (LM and ML) of Landrace and Meishan pigs, the amounts of sex-related CYP mRNAs in the kidney were measured (Fig. 3). Incidentally, the amounts of serum testosterone in 5-month-old male LM and ML pigs, as well as Meishan pigs, were >40 ng/mL, and those in 5-month-old male Landrace pigs were <23 ng/mL.

Fig. 3. Sex Differences in the Amount of CYP mRNAs in the Kidney of 5-Month-Old LM and ML Pigs

The mRNA amount of each CYP in the kidney was determined by real-time RT-PCR and normalized to that of RPL7 as an internal standard. Standard curves were generated using an RT-reaction mixture with total RNA from the kidneys of 5-month-old female Landrace pigs, except for the determination of the amount of CYP2C49 mRNA, where standard curves were generated using an RT-reaction mixture with total RNA from the kidneys of 5-month-old female Meishan pigs. Each circle shows an individual pig (n = 6 and 9 for male and female LM pigs, respectively; n = 7 and 10 for male and female ML pigs, respectively). The bar indicates the average in each group. ^a,b Significant differences between males and females in each breed were assessed by Student’s t-test: ^a p < 0.05, ^b p < 0.01.

In both LM and ML pigs, the mRNA amounts of CYP2A19, CYP2B22 and CYP3A29 in males were much lower than those in females, while the converse was observed for CYP2C33 and CYP4A24/25. For CYP2C49 mRNA, a significant sex difference (males > females) was observed in ML pigs, but no significant difference was observed in LM pigs because of considerable inter-individual differences among male LM pigs. CYP3A46 mRNA was detected in LM and ML pigs, as well as Landrace pigs, and a sex difference (males < females) in the amount of CYP3A46 mRNA was observed in LM and ML pigs but not in Landrace pigs (Table 1, Fig. 1).

Relationship between Amount of Serum Testosterone and Expression of Sex-Related CYP mRNAs in the Kidney

The concentration of serum androgen is thought to be one of the critical factors producing sex and breed differences in the amounts of several CYP mRNAs in the pig kidney. Therefore, the relationship between the serum androgen concentration and the sex-related expression of CYP mRNAs (CYP2A19, CYP2B22, CYP2C33, CYP2C49, CYP3A29, CYP3A46 and CYP4A24/25) in the kidney were further examined using 1-, 3-, and 5-month-old pigs. Some of the 1-month-old pigs (immature, < 10 ng testosterone/mL serum) were treated with TP and used as samples for the present experiments. The concentrations of serum testosterone in the TP-treated pigs were 28–59 ng/mL. In addition, serum testosterone amount increased in an age-dependent manner in males²⁵⁾ but not in females of all the pigs (<7 ng testosterone/mL serum) used. Therefore, as representative samples of females, 1-month-old and 5-month-old pigs but not 3-month-old pigs were used for the present experiments.

The mRNA amounts of CYP2A19, CYP2B22, CYP3A29 and CYP3A46 in all the pigs with high concentrations (> approx. 30 ng/mL) of serum testosterone were low compared with those in all the pigs with low concentrations of serum testosterone (<30 ng/mL). In particular, the amount of CYP3A29 mRNA decreased in a serum testosterone concentration-dependent manner in all the breeds of pig examined (Fig. 4). Furthermore, a serum testosterone concentration-dependent decrease in the amount of CYP3A46 mRNA was also observed in all the pigs except Meishan pigs.

Fig. 4. Negative Correlations between Serum Testosterone Concentrations and Amounts of CYP2A19, CYP2B22, CYP3A29 and CYP3A46 mRNAs in the Pig Kidney

Serum testosterone cencentrations and amounts of CYP mRNA were measured as described in the Materials and Methods. Some of the serum testosterone concentrations of individual pigs were taken from previously obtained data.^24,25) Each symbol represents each individual pig. Open squares and lozenges, 1-month-old males and females, respectively, of Landrace and Meishan pigs (n = 3 for both sexes of Landrace and Meishan pigs); gray squares, 3-month-old male Landrace and Meishan pigs (n = 4 for Landrace pigs and n = 6 for Meishan pigs); closed squares and lozenges, 5-month-old males and females, respectively, of Landrace and Meishan pigs (n = 6 for both sexes of Landrace and Meishan pigs); closed and open triangles, 5-month-old male and female LM pigs, respectively (n = 6 for males and n = 9 for females); closed and open reverse triangles, 5-month-old male and female ML pigs, respectively (n = 7 for males and n = 10 for females); closed and open circles, 1-month-old male and female Landrace and Meishan pigs treated with TP, respectively (n = 3 for both sexes of Landrace and Meishan pigs). The correlations were examined using the data from all the animals tested and determined by regression analysis, where r is the correlation coefficient.

The amounts of CYP2C33 and CYP4A24/25 mRNAs, however, increased in a serum testosterone concentration-dependent manner in all the breeds of pig examined (Fig. 5). The amount of CYP2C49 mRNA increased in a serum testosterone concentration-dependent manner in Meishan, LM and ML pigs but not in Landrace pigs. In addition, little increase in amount of CYP2C49 mRNA was observed in the TP-treated 1-month-old Landrace and Meishan pigs. This suggests that the expression of such TP-associated effect is dependent on the expression level of TP-responsive transcriptional factor(s) for CYP2C49 gene expression, which is involved in maturation.

Fig. 5. Positive Correlations between Serum Testosterone Concentrations and Amounts of CYP2C33, CYP2B49, and CYP4A24/25 mRNAs in the Pig Kidney

Serum testosterone concentrations and the amounts of CYP mRNAs were measured as described in the Materials and Methods. Some of the serum testosterone concentrations of individual pigs were taken from previously obtained data.^24,25) Each symbol represents each individual pig. Open squares and lozenges, 1-month-old males and females, respectively, of Landrace and Meishan pigs (n = 3 for both sexes of Landrace and Meishan pigs); gray squares, 3-month-old male Landrace and Meishan pigs (n = 4 for Landrace pigs and n = 6 for Meishan pigs); closed squares and lozenges, 5-month-old males and females, respectively, of Landrace and Meishan pigs (n = 6 for both sexes of Landrace and Meishan pigs); closed and open triangles, 5-month-old male and female LM pigs, respectively (n = 6 for males and n = 9 for females); closed and open reverse triangles, 5-month-old male and female ML pigs, respectively (n = 7 for males and n = 10 for females); closed and open circles, 1-month-old male and female Landrace and Meishan pigs treated with TP, respectively (n = 3 for both sexes of Landrace and Meishan pigs). The correlations were examined using the data from all the animals tested and determined by regression analysis, where r is the correlation coefficient.

DISCUSSION

In the present study, we initially found sex and breed differences in the amounts of several CYP mRNAs in the kidney using 5-month-old Landrace and Meishan pigs, and their F₁ (LM and ML) progeny. Subsequently, to clarify the effect of androgen on the amounts of renal CYP mRNAs, we examined the effects of castration and/or TP treatment on those amounts. These results are summarized in Table 2. By analyzing such sex and breed differences in relation to the concentration of serum testosterone, we herein demonstrated that serum testosterone is at least one of the critical factors producing those differences in the amount of each CYP mRNA in the kidney.

Table 2. Summary of Androgen-Dependent Sex and Breed Differences in Gene Expression of CYP Isoforms in the Pig Kidney

CYPs	Landrace				Meishan				LM and ML
	Sex differences	CMa)	TP-treatment		Sex differences	CM	TP-treatment		Sex differences
			CM	IFb)			CM	IF
CYP1A1	♂ = ♀	−	−	−	♂ = ♀	−	−	−	−
CYP1A2	♂ = ♀	−	−	−	♂ = ♀	−	−	−	−
CYP2A19	♂ < ♀	↑	↓	↓	♂ < ♀	↑	↓	↓	♂ < ♀
CYP2B22	♂ = ♀	→	↓	↓	♂ < ♀	↑	↓	↓	♂ < ♀
CYP2C33	♂ = ♀	→	↑	↑	♂ > ♀	↓	↑	↑	♂ > ♀
CYP2C49	♂ = ♀	→	→	→	♂>♀	↓	→	↑	♂ > ♀
CYP3A29	♂ < ♀	→	→	↓	♂ < ♀	↑	↓	↓	♂ < ♀
CYP3A46	♂ = ♀	→	↓	↓	ndc)	nd	nd	nd	♂ < ♀
CYP4A24/25	♂ > ♀	↓	↑	↑	♂ > ♀	↓	↑	↑	♂ > ♀
CYP4B1	♂ = ♀	→	→	→	♂ = ♀	→	→	→	−

^a) CM, castrated males; ^b) IF, intact females; ^c) nd, not detected. Symbols, →: not changed; ↑: increase; ↓: decrease; —: not determined.

We previously found sex differences (male < female) in the amounts of CYP1A1 and CYP1A2 mRNAs in the liver of Meishan, LM and ML pigs but not in Landrace pigs.^23–25) However, no such sex differences were observed in the kidney of Landrace and Meishan pigs. On the contrary, there was a sex difference (male > female) in the amount of CYP4A24/25 mRNA in the kidney but not in the liver. Sex differences in the amounts of CYP2A19, CYP2C33 and CYP2C49 mRNAs in the pig kidney, especially in Meishan, LM and ML pigs, correlated with the previous results in the liver^22,25): male < female for CYP2A19; male > female for CYP2C33 and CYP2C49. Interestingly, sex differences (male < female) in the amounts of CYP2B22, CYP3A29 and/or CYP3A46 mRNAs in the kidney of Meishan, LM and ML pigs were the opposite of those previously obtained in the liver.²²⁾ Sex differences in the amount of CYP2A19, CYP3A29 and CYP4A24/25 mRNAs were observed in the kidney of all the breeds of pig examined. Although sex differences in CYP2B22, CYP2C33 and/or CYP2C49 were observed in Meishan, LM and ML pigs, no such sex differences were observed in Landrace pigs.

These sex and breed differences can be explained by the differences in the concentration of serum testosterone in male pigs. Male 5-month-old Meishan, LM and ML pigs had high concentrations of serum testosterone (>approx. 40 ng/mL) while those of Landrace pigs had low concentrations (<23 ng/mL). Because the amount of CYP2A19 and CYP3A29 mRNAs considerably decreased in pigs with > approx. 20 ng/mL of serum testosterone, sex differences (male < female) in their amounts were observed in all the breeds of pig. Likewise, since the amount of CYP4A24/25 mRNA increased in a serum testosterone concentration-dependent manner up to 80 ng/mL, a sex difference (male > female) was observed in all the breeds of pig. However, because clear changes in the amount of CYP2B22, CYP2C33 and CYP2C49 mRNAs occurred at >30 ng/mL of serum testosterone, sex differences in their amounts were observed in Meishan, LM and ML pigs but not in Landrace pigs. Accordingly, such sex and breed differences are thought to be dependent on those of serum testosterone concentration. This hypothesis is further supported by the experiments using castrated and/or TP-treated pigs. Namely, the amounts of those mRNAs in castrated males (<12 ng testosterone/mL in serum) were almost the same as the corresponding females. Serum concentrations of testosterone in 5-month-old castrated males and intact females rose to >100 ng/mL on TP treatment. Consequently, in the TP-treated pigs, the amounts of CYP2C33 and CYP4A24/25 mRNAs, CYP2C33 and CYP4A24/25 mRNAs increased as much as or higher than those in 5-month-old male Meishan pigs, and the amounts of CYP2B22, CYP3A29 and CYP3A46 mRNAs decreased as much as those in the corresponding male pigs.

A difference in the secretion profile of growth hormone (GH) between males and females in many animals, especially in rodents, is thought to result in sex differences in the amounts of several drug-metabolizing enzymes, including CYPs, in the liver.²⁶⁾ For example, sex differences in the mRNA amounts of Ugt, Sult and Cyp4a isoforms in the mouse liver are influenced by male-pattern GH secretion and/or androgen, whereas those sex differences are primarily regulated by androgen in the kidney.^21,27,28) Moreover, a transacting locus, named GH-dependent repression (GDR), is proposed to regulate the gene for female-specific steroid 15α-hydroxylase (Cyp2a4) in the mouse liver,²⁹⁾ but this gene is reported to be reciprocally regulated by androgen in the liver and kidney of male mice.³⁰⁾ Conversely, we have previously found that in Landrace pigs with a sex difference in the GH secretion profile,³¹⁾ there were no sex differences in the hepatic mRNA amounts of several CYP, SULT and UGT isoforms that do show sex differences in Meishan pigs.^22–25,32) Therefore, such sex differences in pigs are thought to be mainly dependent on differences in serum testosterone concentration. However, because the secretion profile of GH is modified by androgen,^33–35) the possibility that the changes in the mRNA amounts observed on castration and/or TP administration occur, at least in part, through an androgen-mediated change in the GH secretion pattern cannot be ruled out.

Breed differences in amounts of the mRNAs of CYP1A1, CYP1A2, CYP2A19, CYP2B22 and CYP4B1 between sex-matched Landrace and Meishan pigs were also observed in the kidney (Table 1), while no such clear breed differences were observed in the liver.^22,25) Additionally, as for CYP2C49 mRNA, individual differences in each breed of pig were observed regardless of serum testosterone concentration, suggesting that these varieties have genetic diversity. Incidentally, pig breeds are not genetically homogeneous. Interestingly, CYP3A46 mRNA was clearly detected in both the kidney and the liver of Landrace pigs but was below the detection limit in both organs of Meishan pigs.²²⁾ Furthermore, CYP3A46 mRNA was clearly detected in both the kidney and the liver of LM and ML pigs, indicating that expression of CYP3A46 mRNA, as well as serum testosterone,²⁵⁾ is determined by autosomal dominant inheritance. Because 5-month-old male LM and ML pigs, but not Landrace pigs, had high concentrations of serum testosterone (>40 ng/mL), a sex difference in the amount of CYP3A46 mRNA was observed in LM and ML pigs but not in Landrace pigs. These findings strongly suggest that genetic (breed-) and tissue-selective factors, together with serum testosterone, are involved in sex- and breed-related expression levels of several CYP genes. Unfortunately, these factors remain unclear.

In conclusion, we herein demonstrate that there are sex and breed differences in the mRNA amounts of CYP2, CYP3, and CYP4 family isoforms in the pig kidney, and strongly suggest that those differences are dependent on genetic host factors, including the concentration of serum androgen. Furthermore, we show tissue differences between the kidney and the liver in sex/androgen-related expression of CYP genes and strongly suggest that there is a tissue-selective factor responsible for the androgen-related expression of CYP genes. Research to identify the sex-related genetic host factor and tissue-selective factor responsible for regulation of the CYP genes is required in the future. In any case, since androgen is one of host factors determining expression levels of several CYPs responsible for drug metabolism, measurement of serum testosterone concentration is considered to contribute to the development of appropriate individual drug treatment.

Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 19K06463, M.K.). The authors thank the Tsukuba Operation Unit 7, Technical Support Center of Central Region, NARO, Tsukuba, Japan for the care of animals and for collecting tissues.

Conflict of Interest

The authors declare no conflict of interest.

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