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BW12C, a potent left-shifting anti-sickling compound in vitro, was administered to normal healthy male Caucasian volunteers. Doses of 2-20 mg kg-1 given by intravenous infusion over 1 h caused a dose-dependent left-shift of the blood-oxygen saturation curve and at the highest dose some 16% of the haemoglobin existed in a high affinity form. Peak left- shift was observed at the end of infusion and decayed thereafter with a mean half-life of approximately 3 h. There were no adverse systemic effects, either clinical, biochemical or haematological, but there was some local irritation at the intravenous infusion site if the infusion was too concentrated. Pharmacokinetic measurements indicated uptake into erythrocytes, low levels in plasma and a volume of distribution not appreciably greater than the blood volume. A pilot radiolabel study indicated extensive metabolism with elimination into the urine. [ABSTRACT FROM AUTHOR]

Background: People with epilepsy may experience an altered pattern of mortality due to cancer. We determined cancer mortality in two distinct populations with epilepsy: (i) an institutionalised cohort with severe epilepsy (SEC) and (ii) another cohort with presumed milder epilepsy (MEC)., Methods: Mortality data from 1977 until 2003 were obtained for the two cohorts (SEC, n=1358 and MEC, n=4494). Standardised mortality ratios (SMRs) for all causes, all cancers and specific cancer sites were calculated., Findings: The SMR for all cancers was significantly elevated in the SEC (SMR: 1.42; 95%CI: 1.18-1.69) but not in the MEC (SMR: 0.93; 95%CI: 0.84-1.03). The SMR for brain and CNS neoplasms was significantly elevated in the MEC; most cancer deaths in younger ages (<45 years) in this cohort were due to brain and CNS neoplasms. There was also a deficit in deaths due to lung cancer (SMR: 0.68; 95%CI: 0.54-0.84) in this cohort. In the SEC, SMRs for oesophageal (SMR: 2.81, 95%CI: 1.35-5.17), liver (SMR: 4.12, 95%CI: 1.12-10.56) and gall bladder (SMR: 7.32, 95%CI: 1.51-21.38) cancers and non-Hodgkin's lymphoma (SMR: 2.67, 95%CI: 0.98-5.80) were elevated., Conclusions: The modest excess of all cancer deaths and large excesses of deaths due to cancers at certain sites in the SEC and the lack thereof in the MEC suggests dissimilarity in the risk factor profile of the two populations. These risk factors, whether life style attributes or antiepileptic drugs, need to be identified.

Nicotinamide, the amide derivative of nicotinic acid, has over the past forty years been given at high doses for a variety of therapeutic applications. It is currently in trial as a potential means of preventing the onset of Type I (insulin-dependent) diabetes mellitus in high-risk, first-degree relatives. Nicotinamide is for regulatory purposes classed as a food additive rather than a drug and has not therefore required the formal safety evaluation normally expected of a new therapy. Because the safety of treatment with megadoses of vitamins cannot be assumed, a full literature review has been undertaken. The therapeutic index of nicotinamide is wide but at very high doses reversible hepatotoxicity has been reported in animals and humans. Minor abnormalities of liver enzymes can infrequently occur at the doses used for diabetes prevention. There is no evidence of teratogenicity from animal studies and nicotinamide is not in itself oncogenic; at very high doses it does however potentiate islet tumour formation in rats treated with streptozotocin or alloxan. There is no evidence of oncogenicity in man. Growth inhibition can occur in rats but growth in children is unaffected. Studies of its effects on glucose kinetics and insulin sensitivity are inconsistent but minor degrees of insulin resistance have been reported. The drug is well tolerated, especially in recent studies which have used relatively pure preparations of the vitamin. Experience to date therefore suggests that the ratio of risk to benefit of long-term nicotinamide treatment would be highly favourable, should the drug prove efficacious in diabetes prevention. High-dose nicotinamide should still, however, be considered as a drug with toxic potential at adult doses in excess of 3 gm/day and unsupervised use should be discouraged.

Paracetamol (acetaminophen) and hydroxyurea were found to inhibit DNA synthesis in a dose-dependent manner in tissue slices in vitro, with little effect on protein synthesis. Considerable variation in the sensitivity of the different tissues was also observed with an order of least sensitive to most sensitive tissue of liver < testis < spleen. The phenolic antioxidant properties of paracetamol are thought to be the mechanism by which paracetamol inhibits DNA synthesis, which led us to study other phenolic antioxidant molecules and flavonoids for specific inhibition of DNA synthesis. (+)-catechin, m-aminophenol, p-aminophenol and p-cresol all displayed a highly specific inhibition of DNA synthesis. Quercetin displayed a preferential inhibition of DNA synthesis but a significant level of inhibition of protein synthesis was also seen. Nordihydroguaiaretic acid (NDGA) and n-propyl gallate showed preferential inhibition of DNA synthesis at the lower doses tested, but at higher doses showed significant inhibition of protein synthesis, presumably because of cytotoxicity. Caffeic acid and naringenin did not display any specific inhibition of DNA synthesis as protein synthesis was equally inhibited at all doses tested. This study demonstrates that certain phenolic antioxidants can inhibit DNA synthesis specifically but this is not a property shared by all phenolic antioxidants; and that these inhibitors show considerable variation in effectiveness between different tissues.

The flux in rat hepatic ratio of adenosine triphosphate levels to adenosine diphosphate levels (ATP/ADP) during the onset and progression of paracetamol-induced cell injury both in vivo and in vitro were investigated and compared. Leakage of lactate dehydrogenase (LDH) and potassium (K+), and mg water/mg dry weight quantified cell injury. ATP and ADP levels were determined using the luciferin-luciferase bioluminescence assay. For in vitro studies, liver slices obtained from phenobarbitone-induced rats were exposed to 10 mM paracetamol for 120 min (T0-T120) and, then incubated without paracetamol up to a further 240 min (T120-T360). For in vivo studies, groups of four phenobarbitone-induced rats received i.p. injections of 800 mg/kg paracetamol. ATP/ADP ratios fall upon exposure to paracetamol both in vitro and in vivo. However, unlike the in vitro situation where the fall in ATP/ADP ratios precedes and accompanies the progression of cell injury, the in vivo fall in ATP/ADP ratios is shown to occur as cell injury measurements begin to recover to control levels. However, despite these differences classic paracetamol-induced centrilobular necrosis is observed to occur both in vitro and in vivo. This study demonstrates that the liver slice model is a simple and useful technique to investigate the underlying mechanisms of paracetamol-induced cell injury.

DNA synthesis in the spleen, testis, thymus, stomach, small intestine and bone marrow was inhibited by 70-90% at 1 h following an oral dose of paracetamol (1 g/kg). This inhibitory effect was still apparent using a lower dose of 125 mg/kg paracetamol, but not when the dose was reduced to 60 mg/kg. In contrast, the liver was resistant to the inhibitory action of paracetamol on DNA synthesis, there being no significant inhibition of DNA synthesis at 500 mg/kg or 1 g/kg paracetamol. These doses and the associated plasma levels are in the range found in human overdose. Tissue levels of paracetamol in the liver, spleen, thymus, kidney and testis were essentially the same as the plasma level. However the apparent paracetamol tissue levels in the stomach wall and duodenum were orders of a magnitude higher than the plasma level. The tissue levels of paracetamol did not explain the differences between tissues in the degree of inhibition of DNA synthesis, in particular the high levels of paracetamol in the tissue of the stomach and duodenum did not result in higher levels of inhibition in these tissues. This study also shows that the inhibitory effect of paracetamol on DNA synthesis is transient. All the tissues, except the spleen, no longer showed inhibition of DNA synthesis by 4 h post paracetamol dosing.

Alcohol elimination was studied in rats of different ages, reproductive states and nutritional deprivation, with the following results: 1) blood levels of ethanol 180 min after a single dose of 1.5 g/kg, ip were significantly higher in adult male (74 days old, N = 5) than in young male rats (34 days old, N = 5): 92.4 +/- 8.4 vs 6.8 +/- 3.4 mg/100 ml, means +/- SD, respectively; 2) when male rats were given a low protein diet for 48 h, blood ethanol levels after a single dose were significantly increased in young males (38.6 +/- 14.6 mg/100 ml) but no effect after a single dose was found in the same animals at an older age (93.2 +/- 5.0 mg/100 ml); 3) blood levels in female rats were higher than in young males both in the virgin and pregnant states, but during lactation a significant drop in blood levels of ethanol was observed. Blood levels of ethanol (mg/100 ml) 180 min after a single dose of 1.5 g/kg, ip, in females, were: virgin (N = 6): 44.9 +/- 16.1, pregnant (N = 5): 40.0 +/- 10.4, lactant (N = 5) 8.8 +/- 5.8. This difference between virgin and pregnant and lactant rats was not related to changes in ADH activity which did not differ between groups. The present study indicates that in male rats the effect of a short-term protein deprivation on ethanol elimination is dependent on the age of the animal. In females, reproductive state is an important factor in determining ethanol elimination.

We have compared the protective effect of fructose in normal Ringer solution during the onset and progression of cell injury induced by paracetamol in rat liver slices with the protective effect of glucose and fructose-1,6-diphosphate. Liver slices obtained from phenobarbitone-induced and non-induced rats were used in a model in vitro system. Slices were exposed to 10 mM paracetamol for 120 min and then incubated without paracetamol in the presence or absence of protective agents for a further 240 min. Cell injury was quantified by measuring leakage of lactate dehydrogenase (LDH) and potassium (K+). Adenosinetriphosphate (ATP) levels were measured using the luciferin-luciferase bioluminescence assay. Addition of higher concentrations of glucose (10-50 mM) to Ringer solution were not found to result in protection at the end of incubation in paracetamol-treated slices obtained from phenobarbitone-induced rats. Neither did sucrose nor mannitol protect. However, exclusion of glucose from Ringer solution resulted in cell injury in paracetamol-treated slices obtained from non-induced rats. Methionine, a known antidote for paracetamol poisoning, failed to protect in this instances but fructose did protect. This suggests that the presence of a glycolytic substrate plays a crucial role in cell protection. Further evidence for this is the finding that iodoacetate, an inhibitor of glycolysis, not only increase cell injury in paracetamol-treated slices but also reverses fructose protection. Fructose-1,6-diphosphate was found to protect against the onset and progression of cell injury in paracetamol-treated slices obtained from phenobarbitone induced rats. This protective agent is found to maintain high ATP levels and cell viability in paracetamol-treated slices at a time when paracetamol-treated slices show a profound loss of ATP levels and a significant increase in cell injury as measured by leakage of LDH and K+.

Fructose protects cells against several types of injury but the mechanism of protection is uncertain. We have used paracetamol injury in rat liver slices as a model system to investigate the role of ATP levels in protection by fructose. Fructose depletes ATP levels in a concentration-dependent fashion in liver slices obtained from non-induced rats. Liver slices recover their ATP levels in the presence of fructose concentrations up to 10 mM. However, in the presence of of 20mM fructose, ATP levels are depleted for the duration of 240 min incubation. Adenine at 100 microM reverses the ATP depletion induced by 20 mM fructose in slices over 240 min incubation. Liver slices obtained from phenobarbitone induced rats were exposed to 10 mM paracetamol for 120 min and, then, incubated without paracetamol, with or without fructose for another 240 min. Introduction of 10 mM or 20 mM fructose in the second stage of incubation prevents paracetamol-induced injury. Fructose at 20 mM induces a rapid and marked depletion in slice ATP levels and these remain low throughout the second 240 min incubation period. Fructose at 10 mM maintains high ATP levels, even in paracetamol-treated slices. There is a profound protective effect against paracetamol-induced injury by either concentration. This suggests that protection is not dependent on high or on low ATP levels. Incubation of paracetamol-treated slices in the presence of 20 mM fructose plus 100 microM adenine in the second 240 min incubation period still results in the same level of protection as with 20 or 10 mM fructose along while reversing the ATP depletion observed with 20 mM fructose.

A fixed combination of the three components, fructose, cyclosporin A and trifluoperazine (FCAT), was found to protect in the late stage of paracetamol-induced liver cell injury both in vivo and in an in vivo/in vitro system. Rats pre-induced with phenobarbitone were given a paracetamol dose of 1 g/kg i.p. The combination of FCAT was given orally 3 h or 3 and 8 h after paracetamol and was able to afford protection as seen by measurements of plasma alanine transaminase (ALT) levels at 24 h. In the in vivo/in vitro system, rats pre-induced with phenobarbitone were dosed with paracetamol 1 g/kg i.p. to initiate injury and liver slices were then taken 3, 4 and 5 h later. The liver slices were then incubated for up to 18 h with the protective agents (FCAT) and the progression of injury followed. Injury was assessed by lactate dehydrogenase (LDH) leakage into the medium and potassium content of the slices. FCAT significantly reduced the injury even as assessed after 5 h in vivo initiation and 18 h progression in vitro. Mitochondrial membrane potential was also maintained in the FCAT-treated liver slices from paracetamol-treated rats as seen by the ability to maintain a gradient of triphenyl methyl phosphonium (TPMP+) between the cell and external medium. All three compounds are required for protection, indicating that more than one event is critical to the survival of the cell and each target point needs to be protected for effective long-term cell survival. The in vivo/in vitro system has been found to give a better comparability to the in vivo situation than injury models that take 6 h or less.

We have investigated the relationship between ATP levels and the onset and progression of cell injury induced by paracetamol overdose both in vivo and in vitro. Liver slices obtained from phenobarbitone-induced and non-induced rats were used in a model in vitro system. Slices were exposed to paracetamol (2-10 mM), for 120 min and then incubated without paracetamol for a further 240 min. ATP levels are reduced upon exposure to paracetamol in liver slices from both phenobarbitone-induced and non-induced rats. Cell injury, as quantified by measuring leakage of lactate dehydrogenase (LDH) and potassium (K+), does not become apparent until 240 min, some 120 min after exposure to paracetamol had ended. This irreversible cell injury is not observed in liver slices from non-induced rats. For in vivo studies rats were phenobarbitone-induced and received i.p. injections of 800 mg/kg body weight paracetamol. Hepatic ATP levels were measured and are found to drop sharply by 3 h post-injection. Development of irreversible hepatic cell injury was assessed by measuring serum enzyme (ALT) activity. ALT levels do not rise until 12 h have elapsed. Paracetamol in overdose gives rise to ATP depletion in liver cells, that is early, independent of paracetamol metabolism and probably spread throughout the lobule. In contrast cell injury is found late and only in our phenobarbitone-induced rats. No cell injury is observed in liver slices from non-induced rats. This suggests that while the level of ATP depletion which is observed may be a necessary part of cell injury by paracetamol, it is not a sufficient cause.

Short-term in vitro methods (2-6 h) for study of cell injury by paracetamol are often used but, in vivo, injury is not apparent until 12 h or later. Many agents which protect in the short-term in vitro systems, such as fructose and glycerol which are effective, even in the late phase, after paracetamol has initiated injury, do not provide any protection in vivo. We have extended the in vitro liver slice system to a more realistic 18 h. Secondly, we have initiated injury with paracetamol in vivo, then followed the progression of injury in an in vitro system. Control liver slices incubated in a HEPES Ringer solution with antibiotics over 18 h show little sign of injury as demonstrated by leakage of lactate dehydrogenase (LDH) into the medium or loss of potassium. Liver slices exposed to 10 mM paracetamol for 2 h in vitro show extensive LDH leak at 6 h which is even more severe at 18 h. Liver slices from animals treated with paracetamol (1 g/kg i.p.) in vivo for 3 h show little LDH leakage at 6 h in vitro but by 18 h injury is very apparent. Fructose and glycerol which protect against paracetamol injury in the short-term (6-h) in vitro system, do not do so when observations are extended to 18 h. They also fail to provide any protection to the slice from animals pre-treated in vivo with paracetamol. Other agents show similar affects. There is no convincing evidence that these short-term protective agents afford any protection in vivo and we show that ibuprofen and dexamethasone do not protect in vivo. It is clear that short-term assays for cell protection have only a limited explanatory value.

Experimental studies in rats have demonstrated that lactating females have blood ethanol levels five times lower than those observed in non-lactating rats. The purpose of the present study was to verify if this phenomenon also occurs in human beings. Five lactating (L) and five control (C) women received, after formal agreement to the experimental procedure, 0.4 g/kg of ethanol as vodka (Stolichnaya, USSR), between 9:00 and 10:15 a.m. Blood and milk samples were collected 10, 20, 40, 60, 90, 150 and 180 min after ethanol ingestion. Ethanol levels in blood and milk were measured by gas chromatography using the head space technique. Results indicated that: time to reach maximal blood levels was significantly longer in the L group (L: 48.0 +/- 10.9, C: 31.2 +/- 16.4 min, means +/- SD), area under the curve was smaller when group L was compared to group C (L: 3821.5 +/- 1240.5, C: 5154.8 +/- 1313.7 mg% x min, means +/- SD), ethanol blood levels (mg/dl) at 150 and 180 min were significantly lower in the L group (150: L, 10.5 +/- 5.6; C, 18.7 +/- 6.8; 180: L, 3.9 +/- 2.8; C, 13.2 +/- 6.4, means +/- SD). Concentration of ethanol in milk was similar to concentration in blood. These results indicate the importance of lactation for ethanol pharmacokinetics and raise questions about the pharmacokinetics of other drugs ingested by lactating women.

The urine of rats fed on 1% paracetamol in the diet for up to 10 weeks was analysed using 500 MHz 1H NMR spectroscopy. After 3 weeks, paracetamol-dosed rats were found to excrete massive quantities of an unknown metabolite in the urine. Using a range of 1 and 2 dimensional 1H NMR spectroscopic techniques, solid phase extraction and mass spectrometry, the metabolite was identified at 5-oxoproline (5OXP, pyroglutamic acid). Rats fed paracetamol plus methionine, which prevents the depletion of sulphur-containing amino acids, did not develop 5OXP-uria during the study period. Quantitative 1H NMR spectroscopy of whole urine showed that no 5OXP appeared in the urine in the first 2 weeks of feeding paracetamol to the animals, but urinary concentrations then rose rapidly up to 1 M in some animals. This unusually high concentration of 5OXP in the urine and its prevention by methionine indicates that chronic high level paracetamol dosing leads to severe depletion of sulphur-containing amino acids including cysteine with consequent disruption of the glutathione cycle.

Ten healthy volunteers were used in two studies investigating the effect of short-term Brassica consumption on caffeine metabolism. In the first study volunteers were given three Brassica-containing meals, the last one 3 h prior to caffeine administration. In the second study volunteers were given two Brassica-containing meals and then fasted overnight before caffeine administration. In both studies the mean plasma half-life of caffeine was reduced by approximately 20% following a Brassica diet, suggesting that Brassica vegetables stimulate caffeine metabolism. When caffeine was given 3 h after the last meal, plasma caffeine concentrations over 6 h, were increased by up to 27% on the Brassica diet compared to controls. This may be due to a transient increased permeability of the intestine to caffeine, immediately following Brassica consumption. This effect was not seen in the second study where there was a 12-h period between the last meal and caffeine administration. There was large interindividual variation in the effect of the Brassica diet on caffeine metabolism.

Nutritional toxicology is now a well established discipline for somatic cells, but no such approach is widely used yet in studies of reproductive toxicology. Reduced dietary intake in mice is known to impair spermatogenesis, and animals in toxicity studies frequently show reduced food intake after dosing. Furthermore, although many human groups have nutritionally inadequate diets, the impact of dietary imbalances on the reproductive system has not been systematically examined. A series of experiments was conducted to dissect the spermatogenic response to dietary alterations in mice and rats. It was found that in mice, the increase in abnormal sperm after such treatment was the result of a lack of calories, while the decrease in sperm counts may have been caused by a lack of protein. In rats, dietary restriction was found only to deplete sperm numbers, probably because of a lack of calories and/or non-energetic components of the diet. Additionally, it was shown that a protein-free diet causes a multiplicity of effects on germ cells, some of which are different in mice and rats. A low-fat diet had an adverse effect on sperm numbers and a similar, but much more pronounced effect was observed in both species fed a carbohydrate-free diet. These alterations of spermatogenic endpoints and the species differences observed, have considerable implications for reproductive toxicology.

The effect of paracetamol on mitochondrial function was studied using rat liver slices. Changes in the potential of the mitochondrial and plasma membrane were monitored using [3H]-triphenylmethylphosphonium (TPMP+) and [14C]thiocyanate (SCN-) probes, respectively. Liver slices were exposed to 10 mM paracetamol for various time periods (0-360 min) after loading with TPMP+. The release of TPMP+ which correlates with a decrease in the mitochondrial membrane potential became significant after 30 min incubation with 10 mM paracetamol. The change in the mitochondrial membrane potential was shown to be independent of cytochrome P450 activity. No significant change in plasma membrane potential was observed, until the release of lactate dehydrogenase (LDH) had begun, 4 hr after exposure, reflecting the ultimate stages of cell injury by paracetamol. These results suggest that paracetamol elicits a direct effect on the mitochondrial function before cell injury develops and adds further evidence to the role of mitochondria in paracetamol toxicity.

Hepatic cell injury was studied in an in vitro system using rat liver slices incubated in two stages. During the first 2 hr slices were exposed to 10 mM paracetamol, this was absent during the subsequent 4 hr of incubation. Cell damage was quantified at the end by measuring leakage of lactic dehydrogenase, increase in water content and potassium loss. Treatment of slices with 20 mM fructose in the second period of incubation prevented paracetamol-induced damage. The effect of fructose was not modified by the continued presence of paracetamol in the second incubation period. The inhibition of glycolysis either with 1 mM NaF or 10 microM iodoacetate blocked the effect of fructose. The protective effect afforded by fructose was not duplicated by the addition of lactate. All these findings strongly suggest an increase in intracellular ATP levels as the most probable explanation for the protective effect of fructose, and point to fructose as a potentially useful therapeutic tool for protection of the liver late in paracetamol intoxication.

The effect of epidermal growth factor (EGF) on sexual maturation of female rats was studied. A within-litter experimental design was employed, so that in each litter each female received four daily injections of EGF (E, 500 ng/g body weight s.c.) or vehicle (V), at one of three ages: days 0-3 (E1, V1), days 8-11 (E2, V2), days 16-19 (E3, V3). Body weight, pinna detachment, incisor eruption, eye opening, auditory startle, visual placing, vaginal opening and first cytological oestrus were assessed. Neonatal treatment with EGF (E1) delayed pinna detachment and the appearance of the auditory startle, but accelerated eye opening. Also, E1, but not E2 and E3, resulted in lower body weight at weaning. Treatment E3 advanced sexual maturation, as indicated by vaginal opening and first cytological oestrus, by 5-6 days. E1 and E2 had no such effect. Hence the sensitive period for the effect of EGF on female rat sexual maturation is later than that for effects on other developmental characteristics and body weight. In a second experiment, ovary and uterus weights were found not to differ between E3 and V3 females killed on the day of vaginal opening of the E3 rats, suggesting that the effect of EGF may be specifically on the perineal epithelium and not on sexual maturation generally.

1. This study was designed to investigate the effect of prolonged paracetamol intake on sulphur metabolism in patients. Six patients, taking paracetamol in doses of 2-4 g d-1 and one taking 12 g d-1 took part in the study. Daily (24-h) collections of urine, and also plasma samples from these patients were analysed and in one case a study with additional methionine was performed. 2. The patients showed variable decrease in urinary output of inorganic sulphate but moderately raised plasma levels of inorganic sulphate. 3. Paracetamol metabolism was characterized by dominance of the glucuronidation pathway with the increase of the administered dose of paracetamol. Methionine added to the diet did not change this metabolic pattern. None of the subjects produced more than 0.6 mmol h-1 paracetamol sulphate while total sulphur excretion was 7.5-26.7 mmol d-1. 4. Volunteers taking 3 g d-1 paracetamol were also studied. 5. In persons taking a normal western diet containing over 20 mmol d-1 sulphur amino-acids paracetamol metabolism will not lead to sulphur amino-acid depletion even if high daily doses are used.

Cell injury by chemicals takes place in two stages, initial chemical interaction between the material and cell components, and subsequent stages of adaptation or failure of response to chemical modification of some parts of the cell apparatus. Paracetamol toxicity was studied in an in vitro system using liver slices incubated in two stages. During the first 2 hr slices were exposed to 10 mM paracetamol and this was absent during the following 4 hr of incubation. Damage was quantified at the end by measuring leakage of lactate dehydrogenase (LDH), increase in water content and potassium loss. Treatment of slices with 10 microM DCPIP or 2 mM ethanol in the second period of incubation prevented paracetamol-induced damage. The protective effect was not dependent on the activity of P450 since the second stage of incubation was insensitive to SKF-525A. The protective effect of ethanol was blocked by the presence of 1 mM pyrazole suggesting that ethanol needs to be metabolized to exert its effect. The addition of 20 mM pyruvate reversed the protection provided by ethanol. Nicotinamide did not alter toxicity or protection by ethanol. These findings suggest an increase of NADH and NADH/NAD ratio as the most probable explanation for the protective effect observed. The role of reducing equivalents in detoxification of quinones such as NAPQI by formation of hydroquinones, is discussed in relation to the mechanism of cell injury by paracetamol.

Chloroacetaldehyde has been shown to be a urinary metabolite of cyclophosphamide in the rat. It was identified in the urine of rats given a mixture of [ring C-4, 14C]-cyclophosphamide and [chloroethyl-3H]-cyclophosphamide as its 2,4-dinitrophenyl-hydrazone derivative by its 3H label and by its co-chromatography with chemically synthesised chloroacetaldehyde. The reaction of chloroacetaldehyde with another urinary metabolite of cyclophosphamide is discussed and a nornitrogen mustard adduct postulated. The toxicity of chloroacetaldehyde is also discussed particularly in relation to the urinary bladder.

Over 2000 epileptic patients admitted to the Chalfont Centre for Epilepsy between 1931 and 1971 and taking anticonvulsants were followed up to the end of 1977. Mortality between 1951 and 1977 was greatly in excess of that in the general population of England and Wales in that period allowing for age and sex. Some of the excess was directly attributable to epilepsy, but there were also more deaths from suicide and circulatory, respiratory, and malignant disease than would be expected. Apart from the brain and central nervous system, no particular site had a significant excess of tumours. In particular, there were no liver tumours (and only one gallbladder carcinoma). This makes it unlikely that the liver tumours produced on feeding phenobarbitone to mice are indicators of major human risk.

Small doses of initiators, such as the nitrosamines, are present in the diet and man is also exposed to promoters, such as phenobarbitone or alcohol. A simple two-stage model of rat hepatocarcinogenesis with a single ip dose of diethylnitrosamine (DEN) as the initiator and sodium phenobarbitone or ethanol given in the drinking-water for 12-18 months as the promoter was used to investigate dose-response relationships for initiation and promotion. Phenobarbitone given alone for 12 months had no carcinogenic effect on rat liver. Low doses of DEN, given prior to phenobarbitone promotion, resulted in the formation of hyperplastic nodules, but only the high dose of DEN (30 mg/kg) resulted in carcinoma formation. Basophilic foci showed a dose-response relationship with DEN and it is suggested that these may be more important than nodules in carcinoma formation and that they represent a heterogeneous group. Only the top concentration of phenobarbitone (1000 micrograms/ml) promoted carcinoma initiated by 30 mg DEN/kg although lower doses of phenobarbitone produce significant enzyme induction. A weak enzyme inducer, 5% ethanol, was as effective a promoter as 1000 micrograms phenobarbitone/ml. Enzyme induction and tumour formation are therefore not directly related. Commencing top-dose phenobarbitone promotion 10 months after the single dose of DEN led to tumour formation, demonstrating the persistence of DEN-initiated cells. The significance to man of the apparent thresholds for tumour initiation and promotion is discussed.

The role of glutathione (GSH) as a determinant of cellular sensitivity to the cytotoxic and DNA-damaging effects of cyclophosphamide (CP) was studied in a dual culture system of rat hepatocytes and K562 human chronic myeloid leukemia cells, which have elevated aldehyde dehydrogenase activity with a corresponding insensitivity to activated CP. Exposure of K562 cells to 50 microM DL-buthionine-S,R-sulfoximine for 24 h resulted in a depletion of cellular GSH content to 10% of control values without toxicity. Subsequent 1-h exposure of GSH-depleted cells to activated cyclophosphamide, obtained by incubation of CP with suspension cultures of rat hepatocytes, resulted in a 5-fold potentiation of the cytotoxicity of CP. Alkaline elution analysis of cellular DNA demonstrated that the level of apparent interstrand cross-linking was 3 to 4 times higher in GSH-depleted cells than in nondepleted cells. GSH-depleted cells were, in addition, more sensitive to induction of DNA single strand breaks than nondepleted cells. Depletion of GSH content did not increase cellular sensitivity to the cytotoxicity of phosphoramide mustard. Preincubation of K562 cells with 1 mM cysteine for 4 h resulted in an approximately 60% increase in cellular GSH content, which was accompanied by decreased sensitivity to the cytotoxicity of hepatocyte-activated CP. Exposure of nondepleted cells to clinically relevant concentrations of hepatocyte-activated CP resulted in depletion of cellular GSH content. Replenishment of GSH content in these cells was relatively slow following CP exposure. Acrolein was highly effective at depleting cellular GSH content, whereas phosphoramide mustard had no effect on cellular GSH content. The depletion of GSH by intracellularly released acrolein may be important in the mechanism of cytotoxicity of CP.

Paracetamol metabolism and toxicity were studied in isolated rat hepatocytes. Cell damage, due to paracetamol, was shown to be dose dependent and was worse in cells from animals pre-treated with phenobarbitone. Exposure to 10 mM paracetamol for 1 hr caused a loss of intracellular reduced glutathione (GSH) and a later progressive leakage of isocitrate dehydrogenase (ICD). Treatment with (+)catechin, 3-O-methyl(+)catechin and promethazine reduced or prevented the paracetamol-induced ICD leakage. Similarly, studies on covalent binding of paracetamol showed that 3-O-methyl(+)catechin, which "protected" the cells, did so without affecting the amount of material bound covalently to cellular protein. Incubation in tissue culture for 24 hr, after prior treatment with paracetamol +/- the protective agent, showed that the protected cells remained viable and attached to tissue culture plates much better than did the "unprotected" cells. These results suggest that the protective effect is much more than just a temporarily delayed cell death. GSH loss and covalent binding of paracetamol metabolites to cell protein are not sufficient causes of cell death, although they may act as starting points in the chain of events leading to cell death.

Rat liver epithelial cells grown in medium without serum show characteristic changes in fatty acid composition and decreased levels of activity of the microsomal enzyme function aryl hydrocarbon hydroxylase. Manipulation of cell fatty acid composition was performed using albumin-fatty acid complexes. Both GLC analysis of fatty acid methyl esters and Arrhenius plots of enzyme activity indicate changes in membrane lipid composition without major changes in aryl hydrocarbon hydroxylase enzyme activity or inducibility by benzanthracene in the presence of butylated hydroxytoluene. When essential fatty acids were added without antioxidant, increased aryl hydrocarbon hydroxylase activity was found. These changes in microsomal aryl hydrocarbon hydroxylase activity were independent of changes in cytochrome c reductase. It is concluded that expression and induction of aryl hydrocarbon hydroxylase is not dependent upon cell fatty acid composition but may be enhanced under conditions of limited lipid peroxidation.

Rats were fed either sodium phenobarbitone (PB) or betanaphthoflavone (BNF) for seven days. Deethylation of 7-ethoxyresorufin ( 7ERR ) and 7-ethoxycoumarin ( 7EC ) was measured in small and large intestine and liver, and cytochrome P-450 in liver. Our semi-purified diet was shown to produce minimal levels of intestinal deethylation activity. BNF was added to the semi purified diet and fed at levels from 0.1 to 100 mg BNF/kg of diet. Significant (P less than 0.05) induction of deethylation in small intestine was seen at all dose levels, ranging from 2-fold at 0.1 mg/kg diet to greater than 100-fold at 100 mg/kg diet. A 3-fold increase was also seen in the large intestine at 50 mg/kg. A significant increase in hepatic deethylation was only seen at 100 mg/kg. PB was administered in drinking water at 50, 100 and 1000 mg PB/l. Significant (P less than 0.05) induction of hepatic deethylation was seen at all dose levels, ranging from 2-fold at 50 mg/l to 5-fold at 1000 mg/l. Hepatic cytochrome P450 was also increased. No significant increase in intestinal deethylation was seen at any of the doses used.

Male Wistar rats were fed a semi-purified diet (MID - minimal inducing diet) with or without addition of 50 ppm of beta-naphthoflavone (BNF) for 1 week. After 1 week the rats were dosed with 20 mg/kg of 1,2-dimethylhydrazine (DMH) subcutaneously and killed at various time intervals from the injection. Enzyme levels were determined in microsomal and cytosolic fractions prepared from the liver and the intestinal mucosa. Feeding of BNF for 1 week caused a 6.5-fold increase of 7-ethoxyresorufin (7-ERR) deethylase in the colon as compared to the controls, but did not alter glutathione (GSH) content nor glutathione-S-transferase (GSHST) activity. Hepatic cytochrome P-450 and 7-ERR deethylation were not significantly altered by feeding of BNF at this concentration, whereas GSH and GSHST were increased by a factor of 1.6 and 2, respectively. In the DMH-dosed rats, O6-methylguanine was formed to a greater extent in the BNF-treated colon than in the controls at 1, 12 and 24 h, whilst N7-methylguanine levels were essentially the same in the induced and uninduced rats. No significant difference was found in the degree of hepatic DNA alkylation at any time points. As shown by our results, the nature of the diet would appear to be able to modulate the rate of metabolic activation of DMH and its binding to DNA in the target organ.

Both white and Savoy-type cabbage added to a semi-purified diet at 25% dry weight and fed to rats ad lib. for 5 days significantly induced ethoxyresorufin (ERR) deethylation in the small and large intestine. Savoy cabbage also induced hepatic activity and, in general, exhibited a greater inducing effect than white cabbage. These enzyme-inducing effects were altered when the cabbage had been processed. The content of intact glucosinolate was greater in Savoy than in white cabbage. The indole glucosinolate (glucobrassicin) content of both types of cabbage was approximately halved by cooking but was unaffected by fermentation, whilst homogenization of Savoy cabbage led to the total disappearance of intact glucosinolates. Levels of the indole glucosinolate breakdown products ascorbigen and indole-3-carbinol were highest in homogenized cabbage, and ascorbigen levels were also higher in cooked than in fresh cabbage of either type. When added to the semi-purified diet and fed ad lib. to rats for 5 days, indole-3-carbinol was a potent inducer of hepatic ERR deethylation and cytochrome P-450 activity, but had much less effect in the intestine. Other glucobrassicin metabolites, diindolylmethane and indole-3-acetonitrile, also had some inducing effect in the liver but no effect in the intestine, while ascorbigen significantly induced ERR deethylation in the small and large intestine but had no effect on hepatic MFO activity.

The in vitro cytotoxicity and mechanism of action of cyclophosphamide (CP) were studied in a dual cell culture system, using rat hepatocytes and K562 human chronic myeloid leukemia cells. Cytotoxicity and DNA damage were measurable in K562 cells using CP concentrations that are clinically attainable. Alkaline elution analysis of cellular DNA demonstrated the presence of concentration- and time-dependent DNA interstrand cross-links, DNA-protein cross-links, and DNA single strand breaks in K562 cells following a 1-h exposure to cyclophosphamide activated by hepatocytes. Hepatocyte-activated CP was 3 to 4 times more potent than phosphoramide mustard with regard to cytotoxicity and induction of DNA interstrand cross-links. Exposure to phosphoramide mustard did not produce single strand breaks, but exposure of K562 cells to acrolein resulted in substantial levels of single strand breaks. The demonstration of acrolein-induced single strand breaks following exposure to activated CP is a novel finding and suggests that acrolein may have a role in the cytotoxicity of CP.

High-performance liquid chromatography (HPLC) with ultraviolet absorption detection, and gas chromatography (GC) with an electron-capture detector have been compared for their convenience in the analysis of ecdysteroids in invertebrate tissues. Analysis by HPLC on reversed-phase materials, including C18, C22 and CN bonded phases, was explored for the separation of both polar ecdysteroids and some of their possible biosynthetic intermediates of lower polarity. The HPLC method was found to be rapid and easy to use, but less sensitive than GC with electron-capture detection. The greater sensitivity and selectivity of GC was found to be important for the more difficult analyses.