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The pharmacokinetics and neurotoxicity of paraquat dichloride (PQ) were assessed following once weekly administration to C57BL/6J male mice by intraperitoneal injection for 1, 2 or 3 weeks at doses of 10, 15 or 25 mg/kg/week. Approximately 0.3% of the administered dose was taken up by the brain and was slowly eliminated, with a half-life of approximately 3 weeks. PQ did not alter the concentration of dopamine (DA), homovanillic acid (HVA) or 3,4-dihydroxyphenylacetic acid (DOPAC), or increase dopamine turnover in the striatum. There was inconsistent stereological evidence of a loss of DA neurons, as identified by chromogenic or fluorescent-tagged antibodies to tyrosine hydroxylase in the substantia nigra pars compacta (SNpc). There was no evidence that PQ induced neuronal degeneration in the SNpc or degenerating neuronal processes in the striatum, as indicated by the absence of uptake of silver stain or reduced immunolabeling of tyrosine-hydroxylase-positive (TH(+)) neurons. There was no evidence of apoptotic cell death, which was evaluated using TUNEL or caspase 3 assays. Microglia (IBA-1 immunoreactivity) and astrocytes (GFAP immunoreactivity) were not activated in PQ-treated mice 4, 8, 16, 24, 48, 96 or 168 h after 1, 2 or 3 doses of PQ. In contrast, mice dosed with the positive control substance, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 10mg/kg/dose×4 doses, 2 h apart), displayed significantly reduced DA and DOPAC concentrations and increased DA turnover in the striatum 7 days after dosing. The number of TH(+) neurons in the SNpc was reduced, and there were increased numbers of degenerating neurons and neuronal processes in the SNpc and striatum. MPTP-mediated cell death was not attributed to apoptosis. MPTP activated microglia and astrocytes within 4 h of the last dose, reaching a peak within 48 h. The microglial response ended by 96 h in the SNpc, but the astrocytic response continued through 168 h in the striatum. These results bring into question previous published stereological studies that report loss of TH(+) neurons in the SNpc of PQ-treated mice. This study also suggests that even if the reduction in TH(+) neurons reported by others occurs in PQ-treated mice, this apparent phenotypic change is unaccompanied by neuronal cell death or by modification of dopamine levels in the striatum.

Historically, toxicology has played a significant role in verifying conclusions drawn on the basis of epidemiological findings. Agents that were suggested to have a role in human diseases have been tested in animals to firmly establish a causative link. Bacterial pathogens are perhaps the oldest examples, and tobacco smoke and lung cancer and asbestos and mesothelioma provide two more recent examples. With the advent of toxicity testing guidelines and protocols, toxicology took on a role that was intended to anticipate or predict potential adverse effects in humans, and epidemiology, in many cases, served a role in verifying or negating these toxicological predictions. The coupled role of epidemiology and toxicology in discerning human health effects by environmental agents is obvious, but there is currently no systematic and transparent way to bring the data and analysis of the two disciplines together in a way that provides a unified view on an adverse causal relationship between an agent and a disease. In working to advance the interaction between the fields of toxicology and epidemiology, we propose here a five-step "Epid-Tox" process that would focus on: (1) collection of all relevant studies, (2) assessment of their quality, (3) evaluation of the weight of evidence, (4) assignment of a scalable conclusion, and (5) placement on a causal relationship grid. The causal relationship grid provides a clear view of how epidemiological and toxicological data intersect, permits straightforward conclusions with regard to a causal relationship between agent and effect, and can show how additional data can influence conclusions of causality.

To predict important strategic issues in product safety during the next 10 years, the Health and Environmental Sciences Institute (HESI) of the International Life Sciences Institute initiated a mapping exercise to evaluate which issues are likely to be of societal, scientific, and regulatory importance to regulatory authorities, the HESI membership, and the scientific community at large. Scientists representing government, academia, and industry participated in the exercise. Societal issues identified include sensitive populations, alternative therapies, public education on the precautionary principle, obesity, and aging world populations. Scientific issues identified include cancer testing, children's health, mixtures and co-exposures, sensitive populations, idiosyncratic reactions, "omics" or bioinformatics, and environmental toxicology. Regulatory issues identified include national and regional legislation on chemical safety, exposure inputs, new technologies, transitioning new science into regulations and guidelines, conservative default factors, data quality, and sensitive populations. Because some issues were identified as important in all three areas (e.g. sensitive populations), a comprehensive approach to assessment and management is needed to ensure consideration of societal, scientific, and regulatory implications. The resulting HESI Combined Challenges Map is not intended to offer a universal description of challenges in safety assessment, nor is it intended to address, advocate, or manage the prioritized issues. Rather, the map focuses on and predicts issues likely to be central to the strategic agendas of individual companies and regulatory authorities in the developed world. Many of these issues will become increasingly important in the future in rapidly developing economies, such as India and China. The scientific mapping exercise has particular value to the toxicology community because it represents the contributions of key scientists from around the world from government, academia, and industry.

The application of genomic technology to toxicology (toxicogenomics) has allowed the simultaneous identification of modified gene expression in response to a toxicant to be established for thousands of mammalian genes. This, together with the development of proteomics, metabonomics and our increasing understanding of individual human polymorphisms, will enable toxicologists in the next century to identify those individuals at particular risk from specific toxins, pesticides and pharmaceutical drugs. However, these new opportunities will prove fruitless unless toxicologists address some of the major issues that presently confront their discipline. If anything, the new technologies impose a greater demand on toxicologists to exercise expert judgement on the meaning of their data, and to apply 'common sense' when balancing risks and benefits.

The comparative uterotrophic responses of ovariectomized Wistar (Han) rats to tamoxifen, toremifene, and 17beta-estradiol have been determined over a period of 72 h. Uterine wet weight; luminal epithelial cell hypertrophy; and BrdU labeling index in the different tissue compartments of the uterus, and the immunohistochemical expression of nuclear estrogen receptor alpha (nERalpha), and nuclear progesterone receptor (nPR) were examined. Luminal epithelial cell hypertrophy was produced by all three compounds to a similar degree. 17beta-Estradiol produced an increase in uterine wet weight due to fluid imbibition over the 3-day period, and an increase in DNA synthesis in the endometrial stromal and myometrial compartments of the uterus, as measured by increased BrdU incorporation. Estradiol increased the expression of nERalpha and nPR in the myometrium with time and decreased nERalpha levels from the overexpressed levels in control ovariectomized rat luminal epithelial cells. Tamoxifen and toremifene caused a smaller increase in uterine weight and the BrdU labeling index in the endometrial stroma and myometrium than did estradiol, and they increased the expression of nERalpha and nPR in the myometrium. Tamoxifen and toremifene differed from estradiol in that they did not decrease the expression of nERalpha in the luminal epithelial cells of the uterus. The response of PR expression was the same for tamoxifen, toremifene, and estradiol, and was therefore considered to be the most reliable indication of an estrogen-agonist effect in this study. The ability to distinguish differential, compartmentalized effects for agonists of estrogen action in the uterus will allow a better risk assessment for new pharmaceuticals that are used as breast cancer chemotherapeutic agents, especially where their use may also be associated with an increased risk of uterine cancers, in particular.

Rats fed a low-protein diet and administered 2-(2-nitro-4-trifluoromethylbenzoyl)cyclohexane-1,3-dione (NTBC) orally at 30 μmol/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 aminotransferase 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 μmol/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.

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.

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.

No Abstract.

The prevalence of asthma and airway hyperresponsiveness (AHR) in highly trained endurance athletes is rising. The type of training (i.e. endurance, or speed and power) seems to influence the airway symptoms. High-intensity exercise and training might contribute to the development of asthma or AHR in athletes previously unaffected by these airway disorders. Repeated hyperventilation of unconditioned air, as well as air containing irritants and/or allergens has been suggested to cause thermal, mechanical, or osmotic airway trauma resulting in damage to the airway epithelium. Subsequent airway inflammatory responses may be responsible for the development of atopy-related symptoms in endurance athletes such as those observed in asthma and AHR. Eosinophils and neutrophils are the inflammatory cells that have been frequently observed to be elevated in the airways of endurance athletes. The trafficking of these cells to the airways may possibly be regulated by TH2 cytokines that are expressed in the airways in response to epithelial cell damage. In addition, these airway inflammatory responses may lead to airway remodelling similar to that which occurs in asthma. The effect of the exercise challenge itself may initiate airway atopy-related and inflammatory responses in endurance athletes. While the literature seems to support the role of local airway conditions and/or events in inducing atopy-related symptoms in athletes, it is proposed that alterations in the hormonal and/or cytokine milieu with intense competition and/or training may also play a role. South African Journal of Sports Medicine Vol. 18 (2) 2006: pp. 46-51

2-(2-Nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione (NTBC) is a potent inhibitor of rat liver 4-hydroxyphenylpyruvate dioxygenase (HPPD) leading to tyrosinemia and corneal opacity. We examined the effect of NTBC on the extent of tyrosinemia and production of corneal lesions in the beagle dog, rabbit and rhesus monkey, as part of safety evaluation on this drug. A single oral dose of 10 mg NTBC/kg to beagle dogs or rabbits increased the concentration of tyrosine in plasma and aqueous humour of the eye, the tyrosinemia being both time- and dose-dependent. Hepatic HPPD was markedly inhibited with little effect on the activity of tyrosine aminotransferase (TAT) and homogentisic acid oxidase at the time of peak plasma tyrosine. Daily oral administration of NTBC to beagle dogs at 0.1, 0.5, 1.5 and 5 mg/kg/day produced corneal opacities with an incidence of 34% following 11 weeks of dosing, which reversed upon withdrawal of the drug. Tyrosine in plasma and aqueous humour was increased at all dose levels, 18 weeks after dosing. In contrast, daily oral administration of NTBC to rabbits for 6 weeks and rhesus monkeys for 12 weeks at 10 mg/kg/day produced no evidence of corneal opacities although tyrosine values were markedly increased. Our studies have shown that NTBC is a potent inhibitor of rabbit, beagle dog and by inference rhesus monkey liver HPPD producing a marked tyrosinemia in all species studied, while only beagle dogs show corneal lesions. The production of corneal lesions in experimental animals exposed to NTBC does not appear to be simply related to the concentration of tyrosine in ocular fluid, other as yet unidentified factors appear to be necessary to trigger tissue injury.

SA Sports Medicine Vol.15(3) 2003: 29-31

Objective. To determine the effect of prolonged endurance exercise on the serum concentrations of eosinophil cationic protein (ECP), immunoglobulin E (IgE) and upper respiratory tract symptoms (URTS). Design. In 11 healthy, experienced volunteers (6 males, 5 females, age 43 ± 9.8 years) the serum concentrations of ECP and IgE were measured, 24 hours prior to projected finishing time, immediately post exercise (IPE), and 3 h, 24 h, and 72 h after an ultramarathon (90 km). Self-reported URTS were also recorded for 14 days after the race. ECP was measured using radioimmunoassay and IgE using the Alastat Microplate Total IgE kit. The after-exercise values were corrected for plasma volume changes, which were calculated from haematocrit and haemoglobin values. Serum concentrations of ECP and IgE were analysed using an analysis of variance (ANOVA) comparing values with before-exercise levels. Level of significance was set at p ≤ 0.05. Results. ECP was significantly elevated at 72 hours (+52%), whilst IgE was not significantly altered after the ultramarathon. There were no reported URTS for the 14 days after the race. Conclusion. The eosinophil is a pro-inflammatory leukocyte involved in bronchial hyperreactivity and allergic inflammation of the airways. IgE is associated with allergic diseases such as asthma and rhinitis. Serum ECP is a sensitive marker of eosinophil activation. The result provides evidence for the non-allergic activation of blood eosinophils during prolonged endurance exercise. Whether this indicates exercise or environmentally induced airway inflammation, or a role for ECP in muscle /tissue repair, are hypotheses that require additional research. SA Sports Medicine Vol.16(2) 2004: 12-16

The US Environmental Protection Agency (EPA) in 1999 issued draft guidelines on carcinogen risk assessment, which included the use mode of action information in the risk assessment process. We have used the five stages of induction of toxicity as described by Aldridge to illustrate in the case of two drugs, tamoxifen and NTBC, how mode of action information played a key role in assessing the risk of cancer and target organ toxicity, respectively.

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.

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.