The molecular basis of perinatal changes occurring in major UDP-glucuronosyltransferase (UGT) family 1 isoforms and in UGT2B1, a relevant isoform belonging to family 2, was analyzed in rat liver. Nonpregnant, pregnant (19–20 days of pregnancy), and two groups of postpartum animals corresponding to early and middle stages of lactation (2–4 and 10–12 days after delivery, respectively) were studied. UGT activity determined in UDP-N-acetylglucosamine-activated microsomes revealed that bilirubin, p-nitrophenol, and ethynylestradiol (17β-OH and 3-OH) but not androsterone and estrone glucuronidation rates, were decreased in pregnant rats. Decreased enzyme activities returned to control values after delivery.p-Nitrophenol, androsterone, and estrone conjugation rate increased in postpartum rats. Western blot analysis performed with anti-peptide-specific (anti-1A1, 1A5, 1A6, and 2B1) antibodies revealed decreased levels of all family 1 isoforms and UGT2B1 during pregnancy. In postpartum animals, protein level recovered (1A5 and 2B1) or even increased (1A1 and 1A6) with respect to control rats. Northern blot analysis suggested that expression of UGT proteins is down-regulated at a post-translational level during pregnancy and that increased levels of 1A1 and 1A6 observed in postpartum rats were associated to increased mRNA. To establish whether prolactin is involved in up-regulation of UGT1A1 and 1A6 postpartum, ovariectomized rats were treated with 300 μg of ovine prolactin per day for 7 days. The data indicated that prolactin was able to increase expression of UGT1A6 (protein and mRNA) but not 1A1. Thus, prolactin is the likely mediator of the increased expression of UGT1A6 observed in maternal liver postpartum.
UDP-glucuronosyltransferases (UGTs) represent a superfamily of enzymes that catalyze the conjugation of glucuronic acid to both endogenous compounds, including bilirubin, bile acids, and steroid and thyroid hormones, and exogenous compounds, including food additives, drugs, and environmental pollutants (for review, see Tephly and Burchell, 1990). Based on the nucleotide and amino acid sequences, UGT isoforms in mammals are grouped into two major families termed 1 and 2. Enzymes belonging to family 1, including a bilirubin cluster (UGT1A1 and UGT1A5) and a phenol cluster (UGT1A6 and UGT1A7), are formed by alternative splicing of an isoform-specific exon encoding a unique N-terminal region with common exons 2 to 5 encoding an identical C-terminal region. In contrast, UGT family 2 isoforms are each derived from an individual gene. The activity of UGT is affected by many factors, including enzyme inducers, aging, diet, diseases, and hormones (for review, see Tephly and Burchell, 1990;Miners and Mackenzie, 1991; Burchell et al., 1994). Regulation may occur either at the gene transcription level, resulting in changes in mRNA and protein levels, or at the level of post-translational processing. Because of association of UGT with the lipid environment (Zakim and Dannenberg, 1992), restriction of the cosubstrate UDP-glucuronic acid (UDPGA) to access the enzyme active site (Berg et al., 1995; Bossuyt and Blanckaert, 1995) and protein-protein interactions derived from oligomer formation (Peters et al., 1984;Ikushiro et al., 1997; Meech and Mackenzie, 1997), enzyme activity toward a specific substrate may also depend on the functional state of UGT.
Many studies have been conducted to determine UGT activity in experimental animals during pregnancy. This subject is of particular interest because changes in metabolizing enzyme activities in maternal liver may change the risk of exposure by the fetus. During pregnancy, significant hormonal changes take place, mainly in sex steroids levels, that may substantially affect disposition of potentially toxic compounds. Most of the studies performed in rats reported a decrease in liver UGT activity (expressed per milligram of microsomal protein) compared with nonpregnant controls, affecting major family 1 substrates such as bilirubin and phenol derivatives and some family 2 substrates such as estradiol (Halac and Sicignano, 1969; Neale and Parke, 1973;Vore and Soliven, 1979; Muraca et al., 1984; Borlakoglu et al., 1993). It is reasonable to speculate that factors, such as those modulating the functional state of UGT, may be affected in such a way that they similarly decrease the activity of all the isoforms tested. We previously reported that substantial changes in microsomal lipid composition and membrane fluidity occur perinatally but, in contrast to what is expected, they positively modulate UGT activity (Luquita et al., 1994). Consequently, a decrease in the expression of UGTs is more likely involved in down-regulation of UGT activities during pregnancy. At present, no studies have been conducted to clarify the molecular basis of UGT regulation during pregnancy.
We reported that hepatic UGT activity toward planar phenols is increased in female rats during lactation, particularly at the late stage of lactation (19–21 days after delivery) (Luquita et al., 1994). Using a polyclonal nonspecific antibody developed against a phenol-conjugating isoform, we detected an increased content of UGT protein in ovariectomized rats in response to ovine prolactin treatment, suggesting that this hormone may be involved in the regulation of UGT expression postpartum (Luquita et al., 1996). We postulated that UGT induction, together with an increased bile secretory function in lactating rats (Liu et al., 1992), may be an adaptive response to increase the biliary excretion of toxic compounds, thus preventing their secretion into breast milk. Taken together, the evidence suggests a differential regulation of UGT in pregnancy and postpartum. Immediately after delivery, levels of progesterone and estrogens are dramatically decreased, and other hormones, i.e., prolactin, are greatly increased. In consequence, differences in UGT activities in maternal liver between pregnant and lactating rats are most reasonably associated with the various hormonal actions. The activity and protein expression of the different UGT isoforms, particularly those involving family 1 substrates, have not been determined in postpartum rats or in response to prolactin administration.
In the present study we analyzed the molecular basis of changes occurring in UGT family 1 isoforms (1A1, 1A5, 1A6, and 1A7) and in UGT2B1, a relevant isoform belonging to family 2, in rat liver at late pregnancy and postpartum. For this purpose, enzyme activity toward the classical substrates of the different isoforms and protein and mRNA levels were systematically determined. The role of prolactin on activity and expression of UGTs from family 1 was also analyzed.
