In the context of massive summer mortality events of the Pacific oyster Crassostrea gigas, the aim of this study was to investigate the early effects on genes, enzymes and haemocyte parameters implicated in immune defence mechanisms in C. gigas oysters exposed to a potentially hostile environment, i.e. to an herbicide alone or within a mixture. Following 2 h of exposure to the herbicide diuron at 1 μg L(-1), the repression of different genes implicated in immune defence mechanisms in the haemocytes and the inhibition of enzyme activities, such as laccase-type phenoloxidase (PO) in the plasma, were observed. The inhibition of superoxide dismutase (SOD) activity in the plasma was also observed after 6 and 24 h of exposure. In the mixture with the herbicides diuron and isoproturon, and the pharmaceutical ibuprofen, catecholase-type PO activity in the plasma and the percentage of phagocytosis in the haemocytes were reduced after 6 h of exposure. Our results showed that early effects on molecular, biochemical and cellular parameters can be detected in the presence of diuron alone or within a mixture, giving an insight of its potential effect in situations that can be found in natural environments, i.e. relatively high concentrations for short periods of time.

Diets high in fat are associated with an increased risk of prostate cancer, although the molecular mechanism is still unknown. We have previously reported that arachidonic acid, an omega-6 fatty acid common in the Western diet, stimulates proliferation of prostate cancer cells through production of the 5-lipoxygenase metabolite, 5-HETE (5-hydroxyeicosatetraenoic acid). We now show that 5-HETE is also a potent survival factor for human prostate cancer cells. These cells constitutively produce 5-HETE in serum-free medium with no added stimulus. Exogenous arachidonate markedly increases the production of 5-HETE. Inhibition of 5-lipoxygenase by MK886 completely blocks 5-HETE production and induces massive apoptosis in both hormone-responsive (LNCaP) and -nonresponsive (PC3) human prostate cancer cells. This cell death is very rapid: cells treated with MK886 showed mitochondrial permeability transition between 30 and 60 min, externalization of phosphatidylserine within 2 hr, and degradation of DNA to nucleosomal subunits beginning within 2-4 hr posttreatment. Cell death was effectively blocked by the thiol antioxidant, N-acetyl-L-cysteine, but not by androgen, a powerful survival factor for prostate cancer cells. Apoptosis was specific for 5-lipoxygenase-programmed cell death was not observed with inhibitors of 12-lipoxygenase, cyclooxygenase, or cytochrome P450 pathways of arachidonic acid metabolism. Exogenous 5-HETE protects these cells from apoptosis induced by 5-lipoxygenase inhibitors, confirming a critical role of 5-lipoxygenase activity in the survival of these cells. These findings provide a possible molecular mechanism by which dietary fat may influence the progression of prostate cancer.

The toxic effects of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) were endoscopically evaluated in several studies conducted between 1975 and 1983 and involving 843 normal volunteers. Anti-inflammatory doses of acetylsalicylic acid (2,400 and 3,900 mg/day) consistently produced significantly more mucosal injury than did any of the newer NSAIDs. Buffering did not reduce the degree of damage. Little or no mucosal injury was seen with placebo,"pro drugs," enteric-coated aspirin, or 1,200 mg/day of ibuprofen (Motrin, Upjohn). However, varying degrees of generally dose-dependent mucosal injury were evident with larger doses of ibuprofen, naproxen, tolmetin sodium, and indomethacin. The amount of mucosal damage after 2,400 mg/day of ibuprofen did not increase when 4,800 mg daily was administered. Duodenal injury corresponded to gastric injury, but it was generally less severe. Short-term studies of one to three days indicated that ibuprofen produced little or no injury when given at a dose of 2,400 mg for one day or 1,600 mg/day for three days. No relation was noted between subjective symptoms and endoscopic findings.

Recent studies have suggested that apoptosis is a key phenomenon in the chemopreventive action of nonsteroidal antiinflammatory drugs (NSAIDs), which exhibit cancer-preventive and tumor-regressive effects in the human colon. The effect of NS-398, N-(2-cyclohexyloxy-4-nitrophenyl)methanesulfonamide, which is a selective inhibitor of cyclooxygenase-2 (COX-2), on the induction of apoptosis in two human colorectal cancer cell lines (Colo320 and THRC) was determined. The apoptotic ratios (-fold vs. control value) of Colo320 in the presence of 100 microM indomethacin and NS-398 were 3.3 +/- 1.5 and 9.0 +/- 0.94, and those of THRC were 2.3 +/- 0.46 and 7.4 +/- 0.87, respectively. The ability of NS-398 to induce apoptosis is greater than that of indomethacin. Both indomethacin and NS-398 reduced the cell proliferation in a concentration-dependent manner. The IC50 values of NS-398 (54.8 +/- 3.6 and 77.2 +/- 4.9 microM) were significantly lower than those of indomethacin (206.3 +/- 43.0 and 180.3 +/- 22.6 microM) at P < 0.01 in Colo320 and THRC cell lines, respectively. These findings suggest that NS-398, a selective inhibitor of COX-2, is a possible candidate for a chemopreventive agent with a potent apoptosis-inducing effect and low ulcerogenic activity.

Recent clinical observations indicate that ibuprofen may alleviate the radiation-induced dysuria that almost invariably occurs during radiation therapy for prostate cancer. Because the use of ibuprofen could consequently become common during radiation therapy for prostate cancer, we have been interested in the potential interactions between ibuprofen and ionizing radiation on prostate tumor cells. The effects of gamma-irradiation and/or ibuprofen on PC3 and DU-145 human prostate carcinoma cells were evaluated in vitro using three model systems. Clonogenic survival was determined by plating cells 24 h after treatment of nearly confluent monolayers. Analysis of cell growth, cell detachment, and apoptotic cell death was carried out over a period of up to 9 days after treatment of PC3 and DU-145 monolayers. The effect of ibuprofen and/or radiation was also probed by observing the inhibition of growth of established PC3 and DU-145 colonies that were treated on the 14th day of colony growth. Ibuprofen enhanced the radiation response of prostate cancer cells in all three in vitro models. Both the cytotoxic and radiosensitizing effects of ibuprofen seem to require concentrations that are higher than those reported to inhibit prostaglandin synthesis, suggesting that other molecular mechanisms may be responsible for ibuprofen cytotoxicity.

A study comparing ibuprofen (600 mg four times a day) vs sulindac (200 mg twice a day), and a placebo in the treatment of painful diabetic peripheral neuropathy was conducted in 18 male outpatients. Discomfort was characterized and rated with a subjective neuropathy score. The response to both ibuprofen and sulindac was better than it was to placebo in the entire group. There were no changes in glucose control or renal function. Further studies are necessary to evaluate the significance of aldose reductase-inhibitor properties of nonsteroidal anti-inflammatory drugs and to select the "best" one of these drugs for the treatment of diabetic neuropathy.

Non-steroidal anti-inflammatory drugs (NS-AIDs) are among the most widely prescribed drugs. In this study, we compared the efficacies of four NSAIDs to inhibit lung tumorigenesis in A/J mice. The tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), was given in drinking water between week 0 and week +7. Groups of 25 mice were fed sulindac (123 mg/kg diet), ibuprofen (263 mg/kg), piroxicam (25 mg/kg) or naproxen (230 mg/kg) in AIN-76A diet from week -2 to the end of the bioassay (week +23). Sulindac was the most effective inhibitor and reduced lung tumor multiplicity by 51%. Ibuprofen and piroxicam reduced lung multiplicity by 38% and 30%, respectively. Naproxen demonstrated no inhibitory capacity. Forestomach tumor multiplicity and incidence were both reduced by sulindac and ibuprofen. Sulindac administered from week -2 to week +7 was less effective (28% inhibition) than when given throughout the bioassay. Sulindac induced more intestinal adhesions than any other NSAID and was directly related to the cumulative dose of sulindac. These results show that chemoprevention of lung tumorigenesis by NSAIDs is not limited to sulindac although it is the most effective.

Nonsteroidal antiinflammatory drugs impair renal function in susceptible patients with cirrhosis and ascites. A new antiinflammatory drug, sulindac, is reported not to affect renal function. To evaluate its renal-sparing mechanism, sulindac was administered for 5 days and ibuprofen for 1 day to 10 patients and paraaminohippurate and inulin clearances, serum and urine eicosanoids, and serum and urine sulindac metabolites were monitored. Ibuprofen reduced renal clearances in the 5 subjects with greatest sodium retention, whereas sulindac had no effect. Plasma concentration of the active sulfide metabolite was markedly increased in liver patients, and this concentration correlated with the inhibition of serum thromboxane (r = 0.75, p = 0.01). The percent inhibition of serum thromboxane with sulindac administration correlated with the inhibition of urinary eicosanoids (r = 0.68-0.81, all p less than 0.02). Ibuprofen was generally a more potent inhibitor of serum and urine eicosanoids. Thus, a major factor in the renal-sparing effect of sulindac appears to be its less potent inhibition of renal and extrarenal cyclooxygenase systems.

Reactive metabolite-modified hepatic protein adducts have been proposed to play important roles in the mechanism(s) of hepatotoxicity of nonsteroidal anti-inflammatory drugs (NSAIDs). In the present study, immunochemical techniques have been used to compare the patterns of drug-protein adducts expressed in livers of mice given single doses of one or other of three different NSAIDs. These were diclofenac and sulindac, which are widely used but potentially hepatotoxic drugs, and ibuprofen, which is considered to be nonhepatotoxic. Specific polyclonal antisera were produced by immunization of rabbits with conjugates prepared by coupling each of the NSAIDs to the carrier protein keyhole limpet hemocyanin. Immunoblotting studies revealed dose-dependent formation of major 110 kDa polypeptide adducts in livers from mice sacrificed 6 h after administration of single doses of either diclofenac (0-300 mg/kg) or sulindac (0-100 mg/kg). Lower levels of several other adducts, of 140 and 200 kDa, were also expressed in livers from these animals. In contrast, livers from mice treated with ibuprofen (0-200 mg/kg) predominantly expressed a 60 kDa adduct and only relatively low levels of a 110 kDa adduct. The various adducts were shown by differential centrifugation to be concentrated in the nuclear fraction of liver homogenates. Those derived from diclofenac and sulindac were further localized, by Percoll density gradient centrifugation, to a subfraction which contained a high activity of the bile canalicular marker enzyme alkaline phosphatase. This suggests that they are concentrated in the bile canalicular domain of hepatocytes. The different patterns of adduct formation raise the possibility that formation of certain NSAID protein adducts, particularly 110 kDa adducts, has toxicological significance.

By analyzing two-dimensional electrophoretic patterns of mouse liver proteins with a computerized image analysis system, we have observed quantitative changes in the abundance of more than 70 proteins in mice treated with various agents. Aroclor 1254, a mixture of polychlorinated biphenyls known to induce a broad spectrum of microsomal activity, induces the largest group of changes (60 proteins altered at p less than 0.001 significance). Phenobarbital produces a small set of characteristic changes that forms part of the much larger Aroclor 1254 effect. Ibuprofen treatment produces a phenobarbital-like pattern of change, with the addition of at least one protein change not observed with any of the other treatments. Cycloheximide and carbon tetrachloride each induces a different characteristic pattern of protein alteration. We have assigned most of the mouse liver proteins to a specific subcellular fraction, and it appears that the predominant class of proteins altered by each compound is present in the soluble phase, rather than in the microsomal fraction. The ability to survey large numbers of tissue proteins for involvement in pharmacologic and toxic effects may allow a more comprehensive understanding of the mechanisms of action in vivo and provide new markers of tissue damage.