Cyclooxygenases (COX) catalyse the oxygenation of arachidonic acid to prostaglandin (PG) endoperoxides. Activity of one of the COX isoforms, COX-2, results in production of prostaglandin E(2) (PGE(2)) via the endoperoxide PGH(2). COX-2 has been implicated in the pathogenesis of colorectal cancer. Malondialdehyde (MDA) is a mutagen produced by spontaneous and enzymatic breakdown of PGH(2). MDA reacts with DNA to form adducts, predominantly the pyrimidopurinone adduct of deoxyguanosine (M(1)G). Here the hypothesis was tested that COX-2 activity in human colon cells results in formation of MDA and generation of M(1)G adducts. M(1)G was detected in basal cultures of human non-malignant colon epithelial (HCEC) and malignant SW48, SW480, HT29 and HCA-7 colon cells, at levels from 77 to 148 adducts/10(8) nucleotides. Only HCA-7 and HT29 cells expressed COX-2 protein. Levels of M(1)G correlated significantly (r = 0.98, P < 0.001) with those of intracellular MDA determined colorimetrically in the four malignant cell types, but neither parameter correlated with expression of COX-2 or PG biosynthesis. Induction of COX-2 expression by phorbol 12-myristate 13-acetate in HCEC cells increased PGE(2) production 20-fold and MDA concentration 3-fold. Selective inhibition of COX-2 activity in HCA-7 cells by NS-398 significantly inhibited PGE(2) production, but altered neither MDA nor M(1)G levels. Malondialdehyde treatment of HCEC cells resulted in a doubling of M(1)G levels. These results show for the first time in human colon cells that COX-2 activity is associated with formation of the endogenous mutagen, MDA. Moreover, they demonstrate the correlation between MDA concentration and M(1)G adduct levels in malignant cells.

Background: Many tumours undergo disregulation of polyamine homeostasis and upregulation of ornithine decarboxylase (ODC) activity, which can promote carcinogenesis. In animal models of colon carcinogenesis, inhibition of ODC activity by difluoromethylornithine (DFMO) has been shown to reduce the number and size of colon adenomas and carcinomas. Indole-3-carbinol (I3C) has shown promising chemopreventive activity against a range of human tumour cell types, but little is known about the effect of this agent on colon cell lines. Here, we investigated whether inhibition of ODC by I3C could contribute to a chemopreventive effect in colon cell lines.

Sepsis is a dysregulated host response to infection that can cause widespread effects on other organs including cardiovascular depression, hypotension and organ failure. The receptor for Nociceptin/Orphanin FQ (N/OFQ), NOP is expressed on immune cells and these cells can release the peptide. Exogenous N/OFQ can dilate blood vessels and this peptide is increased in animal and human sepsis. We hypothesise that NOP receptors are present on vascular endothelial cells and therefore provide the target for released N/OFQ to cause vasodilation and hence hypotension. Using human umbilical vein endothelial cells (HUVEC) and human vascular smooth muscle cells (HVSMC) freshly prepared from umbilical cords and up to passage 4, we assessed NOP mRNA expression by Polymerase Chain Reaction (PCR), NOP surface receptor expression using a fluorescent NOP selective probe (N/OFQ ATTO594 ) and NOP receptor function with N/OFQ stimulated ERK1/2 phosphorylation. As an in vitro sepsis mimic we variably incubated cells with 100ng/ml Lipopolysaccharide and Peptidoglycan G (LPS/PepG). HUVECs express NOP mRNA and this was reduced by ~80% (n = 49) after 24–48 hours treatment with LPS/PepG. Untreated cells do not express surface NOP receptors but when treated with LPS/PepG the reduced mRNA was translated into protein visualised by N/OFQ ATTO594 binding (n = 49). These NOP receptors in treated cells produced an N/OFQ (1μM) driven increase in ERK1/2 phosphorylation (n = 20). One (of 50) HUVEC lines expressed NOP mRNA and receptor protein in the absence of LPS/PepG treatment. In contrast, HVSMC expressed NOP mRNA and surface receptor protein (n = 10) independently of LPS/PepG treatment. These receptors were also coupled to ERK1/2 where N/OFQ (1μM) increased phosphorylation. Collectively these data show that an in vitro sepsis mimic (LPS/PepG) upregulates functional NOP expression in the vascular endothelium. Activation of these endothelial receptors as suggested from in vivo whole animal work may contribute to the hypotensive response seen in sepsis. Moreover, blockade of these receptors might be a useful adjunct in the treatment of sepsis.

Polymicrobial sepsis was induced in either conscious iRhom2 e/e mice (KO) or wildtype (WT) (4 ml/g intraperitoneal caecal slurry 3 ). Eighteen hours later, echocardiography (Vevo3100, VisualSonics, Toronto, Canada under general anaesthesia). TNFa and TNFR1/2 gene transcription were quantified.Male WT (n¼5) mice increased stroke volume 18 h after polymicrobial sepsis (mean difference [95% confidence interval]) (8.0 [3.1 to 12.9] ml; P¼0.004), in contrast to female WT (n¼5) mice (4.2 [e0.7 to e9.1] ml; P¼0.09). In contrast, neither sex in iRhom2 e/e mice (n¼5 each) increased their stroke volume after sepsis (male KO, 4.7 [e0.1 to 9.5] ml; P¼0.06; female KO, 2.2 [e2.6 to 7.0] ml; P¼0.4) (two-way analysis of variance [ANOVA]). Female WT mice had lower ventricular TNFa expression after sepsis, compared with male WTs (mean [SD]) (female 0.01 [0.01] AU; male 0.04 [0.04] AU; P¼0.05). The expression of TNFR1, relative to TNFR2, was upregulated in the female WT compared with male WT (female 9.9 [6.9] AU; male 2.7 [0.8] AU; p¼0.01). In iRHOM2 e/e after sepsis, there was no difference between male and female TNFa expression (male 0.01 [0.01] AU; female 0.03 [0.01] AU; P¼0.5) or TNFR1/TNFR2 expression (male 2.1 [1.0] AU; female 2.9 [0.6] AU; P¼0.9) (two-way ANOVA) (Fig. 4).Sepsis induces higher stroke volume in male, but not female mice, an effect less apparent in iRhom2 e/e mice. TNFa transcription is lower in female WT mice. In iRhom2 e/e mice, where leucocyte derived TNFa is absent, sepsis does not drive a relative hyperdynamic response. These data suggest that sex-specific differences in cardiac molecular physiology determine cardiac physiology in sepsis, rather than leucocytederived mediators.