Safety of high-dose nicotinamide: a review.

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.

Effects of phenolic antioxidants and flavonoids on DNA synthesis in rat liver, spleen, and testis in vitro.

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.

Comparison of paracetamol-induced hepatotoxicity in the rat in vivo with progression of cell injury in vitro in rat liver slices.

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.

Inhibition of DNA synthesis by paracetamol in different tissues of the rat in vivo.

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.

Ethanol elimination by rats as a function of reproductive state, gender and nutritional status.

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.

Ethanol elimination by rats as a function of reproductive state, gender and nutritional status

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 ñ 8.4 vs 6.8 + 3.4 mg/lOO ml, means ñ 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 ñ 14.6 mg/l00 ml) but no effect after a single dose was found in the same animals at an older age (93.2 ñ 5.0 mg/l00 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/l00 ml) 180 min after a single dose of 1.5 g/kg, ip, in females, were: virgin (N=6): 44.9 ñ 16. 1, pregnant (N = 5): 40.0 ñ 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.

Comparison of protection by fructose against paracetamol injury with protection by glucose and fructose-1,6-diphosphate.

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+.

Cell protection by fructose is independent of adenosine triphosphate (ATP) levels in paracetamol injury to rat liver slices.

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.

Protection in the late stages of paracetamol-induced liver cell injury with fructose, cyslosporin A and trifluoperazine.

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.

Adenosine triphosphate (ATP) levels in paracetamol-induced cell injury in the rat in vivo and in vitro.

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.

Cell injury and protection in long-term incubation of liver slices after in vivo initiation with paracetamol: cell injury after in vivo initiation with paracetamol.

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.

Ethanol pharmacokinetics in lactating women.

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.

Ethanol pharmacokinetics in lactating women

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 per cent 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

Induction of 5-oxoprolinuria in the rat following chronic feeding with N-acetyl 4-aminophenol (paracetamol).

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.

The effect of brassica vegetable consumption on caffeine metabolism in humans.

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.

Effects of dietary imbalances on spermatogenesis in CD-1 mice and CD rats.

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.

Effect of paracetamol on mitochondrial membrane function in rat liver slices.

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.