Hennie M.J. Roelofs

The effect of interleukin-1, interleukin-6 and its interrelationship on the synthesis of serum amyloid A and C-reactive protein in primary cultures of adult human hepatocytes

During the acute phase response, synthesis of C-reactive protein and serum amyloid A is increased. To investigate whether the enhanced synthesis of these proteins are due to stimulatory effect of inflammatory mediators such as interleukin-1 (IL-1) and interleukin-6 (IL-6) produced by macrophages and monocytes, primary cultures of adult human hepatocytes were exposed to recombinant (r)IL-1, rIL-6 or rIL-1 and monospecific anti rIL-6 antibodies in the presence of 1 microM dexamethasone. The findings indicate that rIL-1 and rIL-6 both stimulate the liver synthesis of C-reactive protein and serum amyloid A, however monospecific anti rIL-6 antibodies reduce the stimulatory effect of rIL-1 on the synthesis of these proteins. These findings suggest that IL-6 plays a key role in the stimulation of synthesis of serum amyloid A and C-reactive protein by the human liver cells.

Glutathione S-transferase M1 and T1 and cytochrome P4501A1 polymorphisms in relation to the risk for benign and malignant head and neck lesions

Susceptibility to head and neck cancer in a particular individual may depend in part on the metabolic balance between Phase 1 enzymes, such as cytochromes P450 (CYPs), and Phase II enzymes, such as glutathione S‐transferases (GSTs). Genetic variability in CYP and GST isoenzymes may contribute to individual differences in susceptibility to chemical carcinogenesis. GSTM1 and GSTT1 null genotypes as well as polymorphic variants in the CYP1A1 gene that may help determine the risk for head and neck cancer have been described in previous reports.

Tumor necrosis factor (TNF) inhibits interleukin (IL)-1 and/or IL-6 stimulated synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes

Interleukin (IL)-1, IL-6 and tumor necrosis factor (TNF) are considered as important mediators for the modulation of liver synthesis of acute phase proteins. However, studies of the direct effect of individual or a combination of these cytokines on the synthesis of acute phase proteins in human hepatocytes are still very limited. In this study, we have examined the synthesis of C-reactive protein (CRP) and serum amyloid A (SAA) in primary cultures of human hepatocytes exposed to recombinant(r)IL-1 alpha (100 U/ml), rIL-6 (2000 U/ml), rTNF alpha (30 U/ml) and to various combinations of these cytokines in the presence of 1 microM dexamethasone. Monoclonal antibodies to rTNF alpha and monospecific anti-rIL-6 sheep antiserum were also used to investigate the possible endogenous production of TNF or IL-6. The findings indicate: (1) IL-1 and IL-6 are stimulatory cytokines for the liver synthesis of CRP and SAA. Anti IL-6 abolishes the stimulatory effect of IL-1. These findings support the previous observation and indicate that IL-1 exerts its action on the enhanced synthesis of CRP and SAA at least in part via IL-6 production in the liver cell. (2) TNF is an inhibitory cytokine for the liver synthesis of CRP. It inhibits also the stimulatory effect of IL-1 and IL-6 on the synthesis of CRP and SAA. (3) Since anti-TNF enhances the stimulatory effect of IL-6 on the synthesis of CRP and SAA, it seems likely that TNF is also produced by the human hepatocytes. However, further studies for more direct evidence of the liver cell production of TNF, such as the detection of TNF messenger RNA are required.

Over-expression of COX-2 mRNA in colorectal cancer.

BackgroundCyclooxygenase-2 (COX-2, PTGS2) is an enzyme involved in the synthesis of prostaglandins and thromboxanes, which are regulators of biologic processes such as inflammation, cell proliferation and angiogenesis. COX-2 over-expression was reported in many (pre) malignant tissues, but data strongly vary and seem to depend on the methodology used.MethodsNormal colorectal mucosa and paired cancerous tissue from 60 patients with colorectal cancer was investigated for the levels of COX-2 mRNA by real-time quantitative Polymerase Chain Reaction (qPCR). COX-2 levels were expressed relative to either: tissue weight or levels of the housekeeping genes beta-2 microglobulin (B2M) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH).ResultsCOX-2 mRNA levels, normalized with respect to tissue weight or mRNA levels of the housekeeping genes B2M or GAPDH, were over-expressed in 80%, 70% and 40% of the colorectal tumor tissues, as compared to the paired adjacent normal colorectal mucosa samples, respectively. Highest mRNA COX-2 ratios tumor/normal were measured when expressed per mg tissue (mean ratio 21.6). When normalized with respect to the housekeeping genes B2M or GAPDH, mean tumor/normal ratios were 16.1 and 7.5, respectively.ConclusionExpression of COX-2 mRNA levels per mg tissue is most simple in comparison to normalization with respect to the housekeeping genes B2M or GAPDH. Levels of COX-2 mRNA are found over-expressed in almost 80% of the colorectal tumors, compared to paired adjacent normal colorectal mucosa, suggesting a role of COX-2 as a potential biomarker for cancer risk, whereas inhibitors of COX-2 could be of value in chemoprevention of colon cancer.

Liver manipulation causes hepatocyte injury and precedes systemic inflammation in patients undergoing liver resection.

BackgroundLiver failure following liver surgery is caused by an insufficient functioning remnant cell mass. This can be due to insufficient liver volume and can be aggravated by additional cell death during or after surgery. The aim of this study was to elucidate the causes of hepatocellular injury in patients undergoing liver resection.MethodsMarkers of hepatocyte injury (AST, GSTα, and L-FABP) and inflammation (IL-6) were measured in plasma of patients undergoing liver resection with and without intermittent inflow occlusion. To study the separate involvement of the intestines and the liver in systemic L-FABP release, arteriovenous concentration differences for L-FABP were measured.ResultsDuring liver manipulation, liver injury markers increased significantly. Arterial plasma levels and transhepatic and transintestinal concentration gradients of L-FABP indicated that this increase was exclusively due to hepatic and not due to intestinal release. Intermittent hepatic inflow occlusion, anesthesia, and liver transection did not further enhance arterial L-FABP and GSTα levels. Hepatocyte injury was followed by an inflammatory response.ConclusionsThis study shows that liver manipulation is a leading cause of hepatocyte injury during liver surgery. A potential causal relation between liver manipulation and systemic inflammation remains to be established; but since the inflammatory response is apparently initiated early during major abdominal surgery, interventions aimed at reducing postoperative inflammation and related complications should be started early during surgery or beforehand.

A polymorphism in the gene for microsomal epoxide hydrolase is associated with pre-eclampsia

OBJECTIVE Microsomal epoxide hydrolase is an important enzyme involved in the metabolism of endogenous and exogenous toxicants. Polymorphic variants of the human epoxide hydrolase gene vary in enzyme activity. We determined whether genetic variability in the gene encoding for microsomal epoxide hydrolase contributes to individual differences in susceptibility to the development of pre-eclampsia with or without the syndrome of Haemolysis, Elevated Liver enzymes, and Low Platelets (HELLP). METHODS A total of 183 non-pregnant women with a history of pre-eclampsia, 96 of whom had concurrently developed the HELLP syndrome, and 151 healthy female controls were genotyped for the 113Tyr→His polymorphism in exon 3 and the 139His→Arg polymorphism in exon 4 of the epoxide hydrolase gene by a polymerase chain reaction-restriction fragment length polymorphism assay. Chi-square analysis was used for statistical evaluation of differences in polymorphic rates. RESULTS In pre-eclampsia a higher frequency (29%) of the high activity genotype Tyr113 Tyr113 in exon 3 was found as compared to controls (16%, OR 2.0, 95% CI 1.2-3.7). There was no difference between groups for the 139His→Arg polymorphism. In women with a history of pre-eclampsia, no difference in epoxide hydrolase genotypes was found between women who either did or did not develop the HELLP syndrome. In addition, a significant association was found between predicted EPHX activity and pre-eclampsia. CONCLUSIONS Women with the high activity genotype in exon 3, which could reflect differences in metabolic activation of endogenous or exogenous toxic compounds, may have enhanced susceptibility to pre-eclampsia. However, polymorphisms in the epoxide hydrolase gene do not seem to influence the risk for concurrent development of the HELLP syndrome.

Combined polymorphisms in UDP-glucuronosyltransferases 1A1 and 1A6: implications for patients with Gilbert's syndrome

BACKGROUND/AIMS UDP-glucuronosyltransferases (UGTs) are important enzymes involved in glucuronidation of various exogenous and endogenous compounds. Studies were undertaken on the variability of three UGT enzyme activities in human livers. Enzyme activities were associated with genetic polymorphisms in UGT1A1 (UGT1A1*28) and UGT1A6 (UGT1A6*2). UGT1A1*28 is associated with Gilberts syndrome, a deficiency in glucuronidation of bilirubin leading to mild hyperbilirubinemia, whereas UGT1A6*2 may result in low glucuronidation rates of several drugs. METHODS Enzyme activities and genetic polymorphisms were assessed in 39 human liver samples, and polymorphisms were also assessed in blood of 253 healthy controls. RESULTS Associations were found between UGT enzyme activities of bilirubin (B) and 4-nitrophenol (NP; r=0.47, P=0.0024), B and 4-methylumbelliferone (MUB; r=0.54, P=0.0003), and NP and MUB (r=0.89, P<0.0001). In addition to the association between B-UGT enzyme activity and UGT1A1*28 (r=0.45, P=0.0034) as reported earlier, an association between B-UGT and UGT1A6*2 (r=0.43, P=0.007) was found. In 253 Dutch Caucasian controls, co-occurrence of UGT1A1*28 and UGT1A6*2 was found (r=0.9, P<0.0001). CONCLUSIONS Most patients with Gilberts syndrome, in addition to their reduced B-UGT enzyme activity, may have abnormalities in the glucuronidation of aspirin or coumarin- and dopamine-derivatives, due to this combination of UGT1A1*28 and UGT1A6*2 genotypes.

Isoniazid and its toxic metabolite hydrazine induce in vitro pyrazinamide toxicity

Antituberculosis drug-induced hepatotoxicity (ATDH) complicates the treatment of 5-10% of patients treated for active tuberculosis (TB). Knowledge regarding the mechanism of toxicity is still incomplete. Metabolism and the formation of toxic metabolites of the TB drugs may play an important role in the development of ATDH. We studied hepatotoxicity and interactions between isoniazid (INH), its toxic metabolite hydrazine (HYD), rifampicin (RIF) and pyrazinamide (PZA) in human hepatoma cells (HepG2). After 24h pre-treatment with a non-toxic concentration of one of the four compounds, cells were exposed to increasing concentrations of INH, HYD, RIF or PZA. To determine whether pre-treatment increased toxicity, changes in the concentration at which 50% of cell growth was inhibited (IC50) were quantified using the WST-1 cytotoxicity assay. Pre-treatment with INH, HYD or RIF decreased the INH IC50 by 24%, 26% and 15%, respectively, meaning that INH toxicity was increased. INH and HYD pre-treatment decreased the PZA IC50 by 30% and 38%, respectively. HYD and RIF toxicity were not affected by the pre-treatments. The present study is the first to demonstrate that pre-treatment with INH or its toxic metabolite HYD increases the in vitro toxicity of PZA. In addition, pre-treatment with INH, HYD or RIF increases the in vitro toxicity of INH. These results give us greater insight into the development of ATDH.

Glutathione and glutathione S-transferases A1-1 and P1-1 in seminal plasma may play a role in protecting against oxidative damage to spermatozoa

OBJECTIVE To study the levels of glutathione, glutathione S-transferase A1-1, and glutathione S-transferase P1-1 in seminal fluid of fertile and subfertile men. DESIGN Retrospective case-control study. SETTING Departments of gastroenterology, obstetrics and gynecology, and epidemiology and biostatistics in a university medical center. PATIENT(S) Twenty-five subfertile men visiting the fertility clinic and 25 fertile men from midwife practices were recruited. INTERVENTION(S) Collection of semen of subfertile and fertile men. MAIN OUTCOME MEASURE(S) Plasma levels of glutathione and glutathione S-transferases A1-1 and P1-1 in relation to seminal characteristics. RESULT(S) Glutathione, glutathione S-transferase A1-1, as well as glutathione S-transferase P1-1 were found in considerable amounts in seminal fluid of subfertile and fertile men. No differences between groups were found for glutathione S-transferases A1-1 and P1-1. Also, no associations with sperm count, motility, or morphology could be detected. Fertile men had significantly higher glutathione levels as compared with the case of subfertile men. Associations of glutathione with sperm motility quality (r(s) = 0.321) and abnormal sperm morphology (r(s) = -0.496) were found. CONCLUSION(S) The presence of glutathione S-transferases A1-1 and P1-1 in seminal fluid suggests a role in the protection against (oxidative) damage of spermatozoa, whereas glutathione may play a role in male fertility.

Antral glutathione concentration and glutathione S-transferase activity in patients with and without Helicobacter pylori.

Previously we demonstrated an inverse relation between cancer of the gastrointestinal tract and glutathione S-transferase activity of the gastrointestinal mucosa. Chronic infection with H. pylori has been associated with an increased risk of gastric cancer. The aim of this study was to investigate the levels of glutathione and glutathione S-transferase activity in H. pylori-infected and noninfected antral mucosa. Glutathione and glutathione S-transferases were measured in antral biopsies of patients with nonulcer dyspepsia without H. pylori infection (A), with prior H. pylori infection who became H. pylori negative after eradication therapy (B) and with proven H. pylori infection (C). Glutathione concentration and glutathione S-transferase activity in group A were 31.0 (range 6.0–59.6) nmol/mg protein and 810 (range 165–1312) nmol/min/mg protein, in group B 27.0 (range 5.0–53.8) nmol/mg protein and 745 (range 403–1199) nmol/min/mg protein, and in group C 18.5 (range 1.6–55.8) nmol/mg protein and 572 (range 144–1047) nmol/min/mg protein, respectively. The glutathione and glutathione S-transferase values were significantly lower in patients infected with H. pylori than in patients who were H. pylori negative.