Effect of intestinal microflora on the urinary metabolic profile of rats: a (1)H-nuclear magnetic resonance spectroscopy study.

1. Analysis of urine by (1)H-nuclear magnetic resonance (NMR) spectroscopy is used to detect biochemical disturbances predictive of toxicological changes. Recent studies, using (1)H-NMR spectroscopy have suggested that Alderley Park rats can be classified as hippuric acid (HA) or m-(hydroxyphenyl)propionic acid (m-HPPA) excretors. Evidence exists for the role of intestinal microflora in the excretion of aromatic phenolic compounds including HA and m-HPPA. 2. We sought to investigate whether intestinal microflora contribute to the difference in excretion. Urinary HA and m-HPPA levels were monitored to characterize excretion over time. The effect of intestinal microflora on the (1)H-NMR spectrum was also investigated using antibiotics to sterilize the intestine. Finally, the levels of m-HPPA and phenylpropionic acid (a precursor for HA) were analysed in the caecum and colon (entire tissue, including contents). 3. Characterization confirmed the presence of HA and m-HPPA excretors; enquiries revealed that the rats were obtained from two floors within a barriered breeding unit. Housing the rats from the two floors together for 21 days resulted in comparable levels of HA and m-HPPA excretion demonstrating that the profiles are not stable. 4. Following antibiotic treatment, HA and m-HPPA excretion decreased, indicating that intestinal microflora contribute to the excretion of these compounds. Finally, m-HPPA levels were higher in the colon of rats that excreted m-HPPA whilst PPA was increased in the caecum and colon of rats that excreted HA. 5. These results demonstrate that the observed difference in HA/m-HPPA excretion is due to differences in the intestinal microflora.

Pharmacokinetics and pharmacodynamics of NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) and mesotrione, inhibitors of 4-hydroxyphenyl pyruvate dioxygenase (HPPD) following a single dose to healthy male volunteers.

AIMS: NTBC (2-(2-nitro-4-fluoromethylbenzoyl)-1,3-cyclohexanedione) and mesotrione (2-(4-methylsulphonyl-2-nitrobenzoyl)-1,3-cyclohexanedione) are inhibitors of 4-hydroxyphenyl pyruvate dioxygenase (HPPD). NTBC has been successfully used as a treatment for hereditary tyrosinaemia type 1 (HT-1), while mesotrione has been developed as an herbicide. The pharmacokinetics of the two compounds were investigated in healthy male volunteers following single oral administration. The aim of the NTBC study was to assess the bioequivalence of two different formulations and to determine the extent of the induced tyrosinaemia. The mesotrione study was performed to determine the magnitude and duration of the effect on tyrosine catabolism. Additionally, the urinary excretion of unchanged mesotrione was measured to assess the importance of this route of clearance and to help develop a strategy for monitoring occupational exposure. METHODS: A total of 28 volunteers participated in two separate studies with the compounds. In the first study, the relative bioavailability of NTBC from liquid and capsule formulations was compared and the effect on plasma tyrosine concentrations measured. In the second study the pharmacokinetics of mesotrione were determined at three doses. Plasma tyrosine concentrations were monitored and the urinary excretion of mesotrione and tyrosine metabolites was measured. RESULTS: Both compounds were well tolerated at the dose levels studied. Peak plasma concentrations of NTBC were rapidly attained following a single oral dose of 1 mg x kg(-1) body weight of either formulation and the half-life in plasma was approximately 54 h. There were no statistical differences in mean (+/- s.d.) AUC(0,infinity) (capsule 602 +/- 154 vs solution 602 +/- 146 microg x ml(-1) h) or t1/2 (capsule 55 +/- 13 vs solution 54 +/- 8 h) and these parameters supported the bioequivalence of the two formulations. Mesotrione was also rapidly absorbed, with a significant proportion of the dose eliminated unchanged in urine. The plasma half-life was approximately 1 h and was independent of dose and AUC(0,infinity) and Cmax increased linearly with dose. Following administration of 1 mg NTBC x kg(-1) in either formulation, the concentrations of tyrosine in plasma increased to approximately 1100 nmol x ml(-1). Concentrations were still approximately 8 times those of background at 14 days after dosing, but had returned to background levels within 2 months of the second dose. Administration of mesotrione resulted in an increase in tyrosine concentrations which reached a maximum of approximately 300 nmol x ml(-1) following a dose of 4 mg x kg(-1) body weight. Concentrations returned to those of background within 2 days of dosing. Urinary excretion of tyrosine metabolites was increased during the 24 h immediately following a dose of 4 mg mesotrione x kg(-1), but returned to background levels during the following 24 h period. CONCLUSIONS: NTBC and mesotrione are both inhibitors of HPPD, although the magnitude and duration of their effect on tyrosine concentrations are very different. When normalized for dose, the extent of the induced tyrosinaemia after administration of NTBC and over the duration of these studies, was approximately 400 fold greater than that following administration of mesotrione. The persistent and significant effect on HPPD following administration of NTBC make it suitable for the treatment of patients with hereditary tyrosinaemia type 1 (HT-1), whilst the minimal and transient effects of mesotrione minimize the likelihood of a clinical effect in the event of systemic exposure occurring during occupational use.

From toxicological problem to therapeutic use: the discovery of the mode of action of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC), its toxicology and development as a drug.

NTBC is a triketone with herbicidal activity that has been shown to have a novel mode of action by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase in plants. Early studies on the toxicity of this compound found that rats treated with NTBC developed corneal lesions. Investigations aimed at understanding the mechanistic basis for the ocular toxicity discovered that the rats developed tyrosinaemia and excreted large amounts of 4-hydroxyphenylpyruvate and 4-hydroxyphenyllactate, owing to inhibition of the hepatic enzyme 4-hydroxyphenylpyruvate dioxygenase. The corneal lesions resemble those seen when rats are fed a diet supplemented with tyrosine, leading us to conclude that the ocular toxicity seen with NTBC is a consequence of a marked and sustained tyrosinaemia. Studies in collaboration with Professor Sven Lindstedt showed that NTBC was a potent inhibitor of purified human liver 4-hydroxyphenylpyruvate dioxygenase. This interaction lead to the concept of using NTBC to treat patients with tyrosinaemia type 1, to block or reduce the formation of toxic metabolites such as succinylacetoacetate in the liver. Zeneca Agrochemicals and Zeneca Pharmaceuticals made NTBC available for clinical use and, with the approval of the Swedish Medical Products Agency, a seriously ill child with an acute form of tyrosinaemia type 1 was successfully treated in February 1991. Subsequently, other children with this inborn error of metabolism in Sweden and other countries have been treated with NTBC. The drug is now available to those in need via Swedish Orphan AB.

The effect of a low-protein diet and dietary supplementation of threonine on tyrosine and 2-(2-nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione-induced corneal lesions, the extent of tyrosinemia, and the activity of enzymes involved in tyrosine catabolism in the rat.

Rats fed a low-protein diet and administered 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC) orally at 30 mumol/kg/day (10 mg/kg/day) or fed a low-protein diet containing 5 ppm NTBC develop lesions to the cornea of the eye within 3-8 days of exposure with an incidence of about 80%. This treatment also produces a marked inhibition of both hepatic and renal 4-hydroxyphenylpyruvate dioxygenase (HPPD) activity, an induction of hepatic but not renal tyrosine amino transferase activity, and a marked tyrosinemia in the plasma and aqueous humor. The extent of tyrosinemia and changes in the activity of tyrosine catabolic enzymes are similar to those reported for rats fed a normal protein diet and administered NTBC orally at 30 mumol/kg/day. However, the onset of corneal lesions occurs much earlier in rats fed a low-protein diet. The adverse ocular effects of NTBC can be alleviated by supplementing the low-protein diet with 1% w/w threonine. The protection afforded by threonine inclusion in the diet was not due to any amelioration in the extent of inhibition of hepatic HPPD activity or reduction in the extent of the tyrosinemia as measured 8 days after treatment. Rats fed L-tyrosine at 5% w/w in a low-protein diet rapidly develop lesions to the cornea of the eye, which are associated with a marked tyrosinemia, increased hepatic tyrosine aminotransferase activity, and about a 50% reduction in the activity of hepatic HPPD. The onset of corneal lesions produced by feeding a high tyrosine diet could be delayed, but not prevented, by inclusion of 1% w/w threonine in the low-protein diet. The basis for the beneficial effect of dietary supplementation of threonine in alleviating the corneal lesions produced by NTBC is unclear. However, our findings do illustrate that protein deficiency limits the ability of the rat to respond to a tyrosine load produced by inhibition of HPPD.

Tissue distribution of 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1-3-dione (NTBC): effect on enzymes involved in tyrosine catabolism and relevance to ocular toxicity in the rat.

Administration of a single oral dose of 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC) to rats produced a marked tyrosinemia in the plasma and aqueous humor. The tyrosinemia was both time- and dose-dependent with the duration being more marked at the higher doses. The dose-response curve was very steep with a single dose of 1.5 micromol NTBC/kg (0.5 mg/kg) and above producing maximal concentrations of tyrosine in plasma of about 2500 nmol/ml and in aqueous humor of about 3500-4000 nmol/ml at 24 hr after dosing. Analysis of the key hepatic enzymes involved in tyrosine catabolism showed that 4-hydroxyphenylpyruvate dioxygenase (HPPD) was markedly inhibited soon after dosing at either 0.3 or 30 micromol/kg (0.1 or 10 mg/kg) NTBC and that the activity recovered very slowly. In response to the tyrosinemia, the activity of tyrosine aminotransferase (TAT) in the liver was induced about twofold, while the activity of homogentisic acid oxidase (HGO) was not affected. Daily oral administration of NTBC for 6 weeks induced lesions to the cornea of the eye, with a dose of 0.3 micromol/kg/day producing about a 38% incidence and a higher dose of 30 micromol/kg/day a 75% incidence. Administration of a single oral dose of [14C]NTBC at either 0.3 or 30 micromol/kg led to selective retention of radiolabel in the liver and to a lesser extent in the kidneys and the Harderian gland. Concentrations of radioactivity in the liver and kidneys remained constant over 4 days and after the lower NTBC dose were about 2 nmol/g wet wt and 0.9 nmol/g wet wt, respectively. Subcellular fractionation of the liver showed that the majority of the radiolabel, >90%, was associated reversibly with the cytosol fraction. No retention of radiolabel was detected in the cornea, the site of toxicity. Our studies indicate that NTBC binds to protein in rat liver cytosol, inhibits the hepatic cytosolic enzyme HPPD, and causes a marked and sustained tyrosinemia. We suggest that this marked and sustained ocular tyrosinemia produced by NTBC in the rat is responsible for the corneal lesions since similar corneal lesions are produced by feeding rats a high tyrosine diet.

Predicting cancer risk from vinyl chloride exposure with a physiologically based pharmacokinetic model.

A physiologically based pharmacokinetic (PBPK) model capable of describing the metabolism of vinyl chloride (VC) in rats, mice, and humans has been developed and validated by comparison with experimental data from experiments not used in model development. This PBPK model has been used to predict measures of delivered dose (reactive VC metabolites produced in the livers of the affected species) hypothesized to be involved in the induction of liver angiosarcoma in rats, mice, and human populations exposed to VC. Measures of delivered dose in rats were fit to an empirical dose-response model (the linearized multistage model of Crump et al.) and used to make predictions of liver angiosarcoma incidence in mice and human populations exposed to VC. This procedure gave a good prediction of angiosarcoma incidence in mice. Predictions of angiosarcoma incidence in humans were more than two orders of magnitude lower than risk estimations which did not utilize pharmacokinetic data, but were still almost an order of magnitude higher than actually observed in exposed human populations.

Characterization of the interaction of 2-[2-nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6,-tetramethyl- cyclohexane-1,3,5-trione with rat hepatic 4-hydroxyphenylpyruvate dioxygenase.

The synthetic beta-triketones are a novel family of chemicals, developed as herbicides that have activity on grass and broadleaf weeds and are selective in corn. Toxicological evaluation of a number of these chemicals has established that they interfere with rat hepatic tyrosine catabolism in vivo by inhibiting the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD). This paper describes the kinetics of inhibition of rat hepatic HPPD in vitro by the representative beta-triketone 2-[2-nitro-4-(trifluoromethyl)benzoyl]-4,4,6,6- tetramethylcyclohexane-1,3,5-trione (1). A marked inhibition of rat hepatic HPPD was observed when 1 was incubated with the enzyme for 3 min at 37 degrees C prior to the initiation of the enzyme reaction by the addition of substrate. In this system, a concentration of 200 nM 1 resulted in a > 90% loss of HPPD activity, and an apparent IC50 was established at approximately 50 nM. The rate constant for the inactivation of HPPD by 1 was (1.5 +/- 0.2) x 10(-5) s-1 nM-1 as determined by progress curve data of oxygen consumed by HPPD with time. This inhibition is reversible in that the enzyme-inhibitor complex slowly dissociates, with approximately 5.5 +/- 0.6% of the enzyme activity being recovered by 6 h at 25 degrees C (t1/2, 25 degrees C, estimated at 101 +/- 14 h). In short, our studies establish 1 to be a tight-binding inhibitor of rat hepatic HPPD in vitro. This inhibition is characterized by the rapid inactivation of HPPD by the formation of an enzyme-inhibitor complex that dissociates extremely slowly with recovery of enzyme activity.

Inhibition of 4-hydroxyphenylpyruvate dioxygenase by 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione and 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-1,3-dione.

The administration of the compound 2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC) to rats (10 mg/kg body wt) caused an elevation in the concentration of plasma tyrosine and gave products in urine that were identified as 4-hydroxyphenylpyruvate (HPPA) and 4-hydroxyphenyllactate (HPLA). This observed chemically induced tyrosinemia established that this compound perturbs tyrosine catabolism and suggested that the causal effect is the inhibition of 4-hydroxyphenylpyruvate dioxygenase (HPPD). This was confirmed when rat liver HPPD was found to be markedly inhibited by NTBC when the enzyme and chemical were incubated, in vitro, for 3 min at 37 degrees C prior to the initiation of the enzyme reaction by the addition of substrate. At 100 nM NTBC, approximately 90% of the enzyme activity was lost and an IC50 was calculated at approximately 40 nM. The inhibition of HPPD by NTBC (50 nM) is time-dependent; the enzyme activity was reduced by > 50% within 30 sec. Progress curve data of loss of enzyme activity with time gave a rate constant for the inactivation of rat liver HPPD [k*, formation of an HPPD-inhibitor (EI) complex] by NTBC of 9.9 +/- 2.5 x 10(-5) sec-1 nM-1. It was established that NTBC is not irreversibly bound in the EI complex but slowly dissociates with a recovery of enzyme activity of 13.7 +/- 1.0% over a 7-hr period (t1/2, 25 degrees C estimated at 63 hours). In comparison, the compound 2-(2-chloro-4-methanesulfonylbenzoyl)-cyclohexane-1,3-dione (CMBC), an analog of NTBC, gave a similar rate for the inactivation of HPPD (k*, 3.3 +/- 0.8 x 10(-5) sec-1 nM-1), whereas 45 +/- 8% of the enzyme activity was recovered over a 7-hr period (t1/2, 25 degrees C approximately 10 hr). These studies establish that NTBC and CMBC are potent, time-dependent (tight-binding) reversible inhibitors of HPPD. The inhibition is characterized by a rapid inactivation of the enzyme by the formation of an HPPD-inhibitor complex that dissociates with recovery of enzyme activity. In vivo, the inhibition of HPPD causes a tyrosinemia that abates with the recovery of enzyme activity. The understanding of the mechanism by which NTBC perturbs tyrosine catabolism has led to the clinical use of this chemical as the first effective pharmacological therapy for the hereditary disorder tyrosinemia I.

The incorporation of radiolabelled sulphur from captan into protein and its impact on a DNA binding study.

Repeated administration of high doses of captan is known to produce tumours specifically in the duodenum of mice. Captan is not carcinogenic in the rat. In this study, DNA purified from the liver, stomach, duodenum and jejenum of mice dosed with 35S radiolabelled captan was found to contain radioactivity equivalent to Covalent Binding Indices in the range 38-91; that from the bone marrow had a CBI of 2.8. The distribution of radioactivity between the various tissues did not reflect the target organ specificity of captan. Attempts to further purify the DNA samples using caesium chloride gradients resulted in partial separation of the radioactivity from the DNA suggesting that covalent binding to the DNA may not have occurred. A study of the chemical breakdown of captan showed that captan is unstable, producing a variety of potentially reactive species containing sulphur. Evidence was further obtained to show that the sulphur of captan is incorporated into endogenous amino acids and protein. Hepatic DNA from mice dosed with 35S radiolabelled N-acetylcysteine, and two thiazolidine derivatives which are analogous to known metabolites of captan, was radiolabelled to a similar extent to that from captan treated mice. Furthermore, the DNA from each of these treatments had similar properties on caesium chloride gradients. It was concluded that the radioactivity associated with DNA in the captan DNA binding study was present in the low levels of protein which are always associated with purified DNA samples.

Comparison of two methods for determining the toxicokinetics of fluazifop-butyl after intravenous dosing in rats.

1. We have described and compared the use of two blood sampling techniques to measure the kinetics of fluazifop-butyl, a selective herbicide. Following intravenous administration of radiolabelled compound, blood samples were collected from female rats either by tail vein puncture or from chronically implanted catheters inserted in tethered rats. Urine samples were also collected from tethered animals. 2. Both techniques indicate that fluazifop-butyl is rapidly eliminated from blood into urine (t(1/2)3-4.5 h) and the overall blood concentration profiles were comparable between the two methods. However, by using cannulated rats, kinetic data were obtained from individual animals, providing evidence of inter-animal variation and allowing compartmental and statistical analysis. 3. The tethered rat technique is relatively simple and reliable. Compared to tail vein bleeding, results obtained from cannulated animals are more informative, providing comprehensive data from a small number of rats. It is therefore the preferred method for our kinetic based research studies using compounds known to exhibit multicompartmental elimination kinetics.

Macromolecular interactions of inhaled methylene chloride in rats and mice.

The in vivo interaction of methylene chloride and its metabolites with F344 rat and B6C3F1 mouse lung and liver DNA was measured after inhalation exposure to 4000 ppm [14C]methylene chloride for 3 hr. DNA was isolated from the tissues 6, 12, and 24 hr after the start of exposure and analyzed for total radioactivity and the distribution of radioactivity within enzymatically hydrolyzed DNA samples. Covalent binding to hepatic protein was also measured. A further group of rats and mice were dosed intravenously with [14C]formate after exposure to nonradiolabeled methylene chloride for 3 hr to determine the pattern of labeling resulting from incorporation of formate into DNA via the C-1 pool. Low levels of radioactivity were found in DNA from lungs and livers of both rats and mice exposed to [14C]methylene chloride. Two- to fourfold higher levels were found in mouse DNA and protein than in rat. Chromatographic analysis of the DNA nucleosides showed the radioactivity to be associated with the normal constituents of DNA. No peaks of radioactivity were found that did not coincide with peaks of radioactivity present in hydrolyzed DNA from formate-treated rats and mice. Under the conditions of this study there was no evidence for alkylation of DNA by methylene chloride in either rats or mice.

Species differences in response to trichloroethylene. I. Pharmacokinetics in rats and mice.

The elimination of radioactivity in two strains of rats and mice following a single po dose of trichloro[14C]ethylene at dose levels from 10 to 2000 mg/kg has shown a marked dose dependence in rats but not in mice. The metabolism of trichloroethylene in the mouse was linear over the range of doses used, whereas in the rat it became constant and independent of dose at 1000 mg/kg and above. At the 10-mg/kg dosage, both species metabolized trichloroethylene almost completely, 60% of the dose being excreted in urine with only 1 to 4% being eliminated unchanged in expired air in the first 24 hr. At 2000 mg/kg, 78% of the dose was eliminated unchanged in the rat, but only 14% in the mouse. Consequently at high dosages, the mouse was exposed to significantly higher concentrations of trichloroethylene metabolites than the rat. Blood level kinetics of trichloroethylene and its metabolites confirmed a faster rate of metabolism in the mouse than in the rat. Peak concentrations of the metabolites were reached within 2 hr of dosing in the mouse compared to 10 to 12 hr in the rat. The concentrations of both trichloroethanol (4X) and trichloroacetic acid (7X) were significantly higher in the mouse than in the rat. Whereas trichloroethanol was rapidly eliminated from blood, the higher concentrations of trichloroacetic acid were maintained for over 30 hr. The high blood quantities of trichloroethylene-derived trichloroacetic acid are known to induce hepatic peroxisome proliferation in mice but are insufficient to induce this response in rats. These data suggest that trichloroacetic acid blood amounts, peroxisome proliferation, and the link between peroxisomes and liver cancer are the basis of species difference in response to trichloroethylene.