Georgette Howard

Activities of conjugating and antioxidant enzymes following endotoxin exposure

Endotoxin exposure elicits various responses in mammals including the acute phase response that has been shown to cause changes in the activity of several forms of cytochrome P450s and other enzymes. Therefore, the hepatic conjugating enzyme, glutathione S‐transferase (GST), and UDP‐glucuronosyltransferase (UDPGT), the antioxidant enzymes, glutathione peroxidase (GSHPx), catalase, and superoxide dismutase (SOD), as well as lipid peroxidation were investigated following the administration of endotoxin to male Sprague–Dawley rats (8 mg/kg body weight). Rats were euthanized at various times following endotoxin administration and the livers removed and processed to assess various enzyme activities. Glutathione S‐transferase, UDPGT, and GSHPx activity showed statistically significant decreases after 24 hours and remained lower than controls for the duration of the study. Decreases in total SOD and catalase activities were seen at 24, 48, and 72 hours following endotoxin administration; however, only catalase activity showed statistically significant differences between control and treated samples at those time points, and total SOD activity showed a statistically significant decrease at 24 hours. No statistically significant changes were seen in the level of lipid peroxidation in the liver microsomes from endotoxin‐treated animals. Changes in the conjugative enzymes and the free‐radical scavenging enzymes following endotoxin exposure may alter the hosts metabolism and response to free radicals.

The effect of high dose endotoxin on CYP3A2 expression in the rat.

AbstractPurpose. The purpose of our research was two-fold: 1) to further characterize the downregulation of CYP3A2 mRNA, protein, and activity during an acute phase response (APR); 2) most importantly, to relate the time-dependent activation of nuclear proteins to putative DNA binding sequences within the CYP3A2 5′-flanking region, with the loss in CYP3A2 expression. Methods. Rats were injected (2.0 mg/animal, i.p.) with LPS and sacrificed at 1,2, 4, 6, 8, 24, 48, and 72 hours. Hepatic nuclear protein was isolated and analyzed for binding activity to AP-1, NFκB, and NF-IL6 consensus sequences. Hepatic CYP3A2 mRNA levels were determined by solution hybridization and CYP3A2 protein, CYP3A2 activity, and total P450 were measured in hepatic microsomes. Results. Computer analysis of the 5′-flanking region of CYP3A2 revealed the presence of 5 NF-IL6 and 4 AP-1 putative DNA binding sites. The strongest increase in AP-1 binding activity occurred between 6 and 24 hr, and the alteration in binding complexes to an NF-IL6 oligonucleotide occurred between 4 and 24 hr. Maximum loss in CYP3A2 mRNA occurred at 8 hr post-LPS injection and remained lowered at the 24 hr timepoint. CYP3A2 protein was significantly decreased at 24, 48, and 72 hours post-LPS treatment with corresponding decreases in CYP3A2 activity and total P450. Conclusions. The changes in NF-IL6 and AP-1 binding after LPS treatment, which appears to correlate with the changes in CYP3A2 mRNA, combined with the presence of putative NF-IL6 and AP-1 sites located in the CYP3A2 5′-flanking region, may indicate a potential role for NF-IL6 and AP-1 in CYP3A2 downregulation during an APR.

The effect of endotoxin on hepatocyte nuclear factor 1 nuclear protein binding: potential implications on CYP2E1 expression in the rat.

The purpose of this study was to determine if changes in nuclear protein binding of hepatocyte nuclear factor 1 (HNF‐1) occur after lipopolysaccharide (LPS) administration. In addition, the time‐course of alterations in CYP2E1 regulation were evaluated. Rats were injected with 2.0 mg LPS and euthanized over a 72‐h period. Nuclear protein binding to a consensus HNF‐1 oligonucleotide was assessed by the electrophoretic mobility shift assay. CYP2E1 activity was analysed using chlorzoxazone as a substrate (6OH‐CLZ), and CYP2E1 protein concentration was determined by enzyme‐linked immunosorbent assay. Endotoxin treatment resulted in decreased nuclear protein binding to an HNF‐1 element as early as 1 h after treatment and returned to control levels by 72 h. This reduced binding persisted for 24 h and returned to control values 48 h after LPS administration. In addition, the reduction in binding was primarily attributable to a HNF‐1α immunoreactive protein. The observed reduction in HNF‐1 binding was followed in the time‐course by decreases in CYP2E1 activity and protein content with maximal decreases to 50 and 67% of control, respectively, at 48 h after LPS administration. Endotoxin is a potent inducer of the acute phase response (APR). The APR stimulation by endotoxin administration reduced HNF‐1α binding and decreased the expression of CYP2E1 in the rat liver. The time‐course of alterations in HNF‐1 and CYP2E1 lend support to the possibility that HNF‐1α may play a role in the down‐regulation of genes that require HNF‐1α for their constitutive expression. These data serve as an important precedent for future studies evaluating the direct association of decreased HNF‐1α binding and reduced gene expression after LPS administration.

Dopaminergic Regulation of Galanin Gene Expression in the Rat Anterior Pituitary Gland

Estrogen dramatically increases galanin mRNA and peptide levels in the rat anterior pituitary gland. We recently reported that galanin secretion in vitro from estrogen‐exposed anterior pituitary cells is regulated by hypothalamic factors; dopamine and somatostatin inhibit galanin secretion, and thyrotrophin‐releasing hormone stimulates galanin release. To determine whether galanin is regulated by a dopaminergic mechanism in vivo, we used ovariectomized Fischer 344 rats treated with 17ß‐estradiol‐containing or empty Silastic capsules. Rats were also administered bromocriptine, a dopamine receptor agonist, haloperidol, a dopamine receptor antagonist, or placebo for 2 weeks. Galanin peptide levels were measured in the anterior pituitary, neurointermediate lobe, medial basal hypothalamus, and plasma by radioimmunoassay. Plasma and pituitary prolactin levels were also determined. Bromocriptine decreased gaianin peptide levels in the anterior pituitary gland of ovariectomized rats by 30%, but had no effect on galanin in the neurointermediate lobe or medial basal hypothalamus. In contrast, haloperidol had no effect on galanin in the anterior pituitary or medial basal hypothalamus of ovariectomized rats, but decreased galanin peptide levels in the neurointermediate lobe. In the anterior pituitary gland of estrogen‐treated rats, bromocriptine increased and haloperidol decreased both galanin and prolactin levels. Galanin mRNA levels were quantified in the anterior pituitary gland by solution hybridization. Bromocriptine increased galanin mRNA levels 3‐fold in the anterior pituitary, whereas haloperidol had no effect. Galanin mRNA levels in the anterior pituitary were elevated 10‐fold by estrogen. Bromocriptine reduced galanin mRNA levels in the pituitary by 50% in estrogen‐treated rats, where again haloperidol had no effect. Estrogen increased plasma galanin levels 4‐fold compared to ovariectomized rats and this effect was reduced 60% by bromocriptine and increased 20% by haloperidol.

Characterization of nuclear protein binding (AP-1, GR, and STAT) in the genetically obese (fa/fa) Zucker rat.

There is evidence to suggest that obese populations have an increased susceptibility to various pathologic disorders. Both AP-1 and STAT nuclear binding proteins have been suggested to play a role in certain obesity-related diseases. The objective of our studies reported herein was to compare constitutive binding activity of nuclear proteins (AP-1, GR, and STAT), that may be relevant to obesity-related diseases in the obese (fa/fa) Zucker rat to lean (Fa/?) littermates. AP-1, GR, and STAT liver nuclear protein binding activity was analyzed using the electrophoretic mobility shift assay (EMSA). EMSA analysis of liver nuclear protein from obese and lean Zucker rats revealed high constitutive AP-1 binding activity in the obese animals. AP-1 binding activity in the obese rats was not further elevated by treatment with phenobarbital, a known inducer of AP-1 binding activity. No differences were observed in GR binding to a consensus GRE between obese and lean animals; however, STAT binding activity to a consensus GAS element was lower in liver tissue from obese Zucker rats. Our findings presented herein suggest that the fa/fa Zucker rat may be a suitable obese rodent model for studying the roles AP-1 and STAT may play in the pathologies of these diseases.