The importance of pyruvate availability to PDC activation and anaplerosis in human skeletal muscle.
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No studies have singularly investigated the relationship between pyruvate availability, pyruvate dehydrogenase complex (PDC) activation, and anaplerosis in skeletal muscle. This is surprising given the functional importance attributed to these processes in normal and disease states. We investigated the effects of changing pyruvate availability with dichloroacetate (DCA), epinephrine, and pyruvate infusions on PDC activation and accumulation of acetyl groups and tricarboxylic acid (TCA) cycle intermediates (TCAI) in human muscle. DCA increased resting PDC activity sixfold (P < 0.05) but decreased the muscle TCAI pool (mmol/kg dry muscle) from 1.174 +/- 0.042 to 0.747 +/- 0.055 (P < 0.05). This was probably a result of pyruvate being diverted to acetyl-CoA and acetylcarnitine after near-maximal activation of PDC by DCA. Conversely, neither epinephrine nor pyruvate activated PDC. However, both increased the TCAI pool (1.128 +/- 0.076 to 1.614 +/- 0.188, P < 0.05 and 1.098 +/- 0.059 to 1.385 +/- 0.114, P < 0.05, respectively) by providing a readily available pool of pyruvate for anaplerosis. These data support the hypothesis that TCAI pool expansion is principally a reflection of increased muscle pyruvate availability and, together with our previous work (J. A. Timmons, S. M. Poucher, D. Constantin-Teodosiu, V. Worrall, I. A. Macdonald, and P. L. Greenhaff. J. Clin. Invest. 97: 879-883, 1996), indicate that TCA cycle expansion may be of little functional significance to TCA cycle flux. It would appear therefore that the primary effect of DCA on oxidative ATP provision is to provide a readily available pool of acetyl groups to the TCA cycle at the onset of exercise rather than increasing TCA cycle flux by expanding the TCAI pool. (+info)
Drinking water disinfection byproducts: review and approach to toxicity evaluation.
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There is widespread potential for human exposure to disinfection byproducts (DBPs) in drinking water because everyone drinks, bathes, cooks, and cleans with water. The need for clean and safe water led the U.S. Congress to pass the Safe Drinking Water Act more than 20 years ago in 1974. In 1976, chloroform, a trihalomethane (THM) and a principal DBP, was shown to be carcinogenic in rodents. This prompted the U.S. Environmental Protection Agency (U.S. EPA) in 1979 to develop a drinking water rule that would provide guidance on the levels of THMs allowed in drinking water. Further concern was raised by epidemiology studies suggesting a weak association between the consumption of chlorinated drinking water and the occurrence of bladder, colon, and rectal cancer. In 1992 the U.S. EPA initiated a negotiated rulemaking to evaluate the need for additional controls for microbial pathogens and DBPs. The goal was to develop an approach that would reduce the level of exposure from disinfectants and DBPs without undermining the control of microbial pathogens. The product of these deliberations was a proposed stage 1 DBP rule. It was agreed that additional information was necessary on how to optimize the use of disinfectants while maintaining control of pathogens before further controls to reduce exposure beyond stage 1 were warranted. In response to this need, the U.S. EPA developed a 5-year research plan to support the development of the longer term rules to control microbial pathogens and DBPs. A considerable body of toxicologic data has been developed on DBPs that occur in the drinking water, but the main emphasis has been on THMs. Given the complexity of the problem and the need for additional data to support the drinking water DBP rules, the U.S. EPA, the National Institute of Environmental Health Sciences, and the U.S. Army are working together to develop a comprehensive biologic and mechanistic DBP database. Selected DBPs will be tested using 2-year toxicity and carcinogenicity studies in standard rodent models; transgenic mouse models and small fish models; in vitro mechanistic and toxicokinetic studies; and reproductive, immunotoxicity, and developmental studies. The goal is to create a toxicity database that reflects a wide range of DBPs resulting from different disinfection practices. This paper describes the approach developed by these agencies to provide the information needed to make scientifically based regulatory decisions. (+info)
Effects of dichloroacetate infusion on human skeletal muscle metabolism at the onset of exercise.
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This study investigated whether dichloroacetate (DCA) decreases the reliance on substrate level phosphorylation during the transition from rest to moderate-intensity exercise in humans. Nine subjects cycled at approximately 65% of maximal oxygen uptake (VO(2 max)) after a saline or DCA (100 mg/kg body wt) infusion, with muscle biopsies taken at rest and at 30 s and 2 and 10 min of exercise. DCA infusion increased pyruvate dehydrogenase (PDH) activation at rest (4.0 +/- 0.3 vs. 0.9 +/- 0.1 mmol. kg wet wt(-1). min(-1)) and at 30 s (3.6 +/- 0.2 vs. 2.5 +/- 0.4 mmol. kg(-1). min(-1)) of exercise. As a result, acetyl-CoA (45.9 +/- 5.9 vs. 11.3 +/- 1.5 micromol/kg dry wt) and acetylcarnitine (13.1 +/- 1.0 vs. 1.6 +/- 0.3 mmol/kg dry wt) were markedly increased by DCA infusion at rest. These differences were maintained at 30 s and 2 min for both acetyl-CoA and acetylcarnitine. Resting muscle lactate and phosphocreatine (PCr) were not different between trials, but DCA infusion resulted in lower lactate accumulation throughout exercise, especially at 2 min (21.6 +/- 3.1 vs. 44.6 +/- 8.0 mmol/kg dry wt). PCr utilization in the initial 30 s (16.9 +/- 0.4 vs. 31.7 +/- 2.6 mmol/kg dry wt) and 2 min (27.8 +/- 4.7 vs. 45.1 +/- 2.6 mmol/kg dry wt) of exercise was decreased with DCA. This resulted in a lower accumulation of free inorganic phosphate at 30 s (25.4 +/- 2.0 vs. 36.4 +/- 2.8 mmol/kg dry wt) and 2 min (34.6 +/- 4.7 vs. 50.5 +/- 2.2 mmol/kg dry wt) with DCA and decreased glycogenolysis over 10 min. The data from this study support the hypothesis that increased provision of substrate by DCA infusion increases oxidative metabolism during the rest-to-work transition, resulting in decreased PCr utilization and an improved cellular energy state at the onset of exercise. The transitory improvement in energy state decreased glycogenolysis and lactate accumulation during moderate-intensity exercise. (+info)
PDH activation by dichloroacetate reduces TCA cycle intermediates at rest but not during exercise in humans.
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We hypothesized that dichloroacetate (DCA), which stimulates the pyruvate dehydrogenase complex (PDH), would attenuate the increase in muscle tricarboxylic acid cycle intermediates (TCAI) during exercise by increasing the oxidative disposal of pyruvate and attenuating the flux through anaplerotic pathways. Six subjects were infused with either saline (Con) or DCA (100 mg/kg body mass) and then performed a moderate leg kicking exercise for 15 min, followed immediately by intense exercise until exhaustion (Exh; approximately 4 min). Resting active fraction of PDH (PDH(a)) was markedly increased (P +info)
Skeletal muscle metabolism during high-intensity sprint exercise is unaffected by dichloroacetate or acetate infusion.
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This study investigated whether increased provision of oxidative substrate would reduce the reliance on nonoxidative ATP production and/or increase power output during maximal sprint exercise. The provision of oxidative substrate was increased at the onset of exercise by the infusion of acetate (AC; increased resting acetylcarnitine) or dichloroacetate [DCA; increased acetylcarnitine and greater activation of pyruvate dehydrogeanse (PDH-a)]. Subjects performed 10 s of maximal cycling on an isokinetic ergometer on three occasions after either DCA, AC, or saline (Con) infusion. Resting PDH-a with DCA was increased significantly over AC and Con trials (3.58 +/- 0.4 vs. 0.52 +/- 0.1 and 0.74 +/- 0.1 mmol. kg wet muscle(-1). min(-1)). DCA and AC significantly increased resting acetyl-CoA (35.2 +/- 4.4 and 22.7 +/- 2.9 vs. 10.2 +/- 1.3 micromol/kg dry muscle) and acetylcarnitine (12.9 +/- 1.4 and 11.0 +/- 1.0 vs. 3.3 +/- 0.6 mmol/kg dry muscle) over Con. Resting contents of phosphocreatine, lactate, ATP, and glycolytic intermediates were not different among trials. Average power output and total work done were not different among the three 10-s sprint trials. Postexercise, PDH-a in AC and Con trials had increased significantly but was still significantly lower than in DCA trial. Acetyl-CoA did not increase in any trial, whereas acetylcarnitine increased significantly only in DCA. Exercise caused identical decreases in ATP and phosphocreatine and identical increases in lactate, pyruvate, and glycolytic intermediates in all trials. These data suggest that there is an inability to utilize extra oxidative substrate (from either stored acetylcarnitine or increased PDH-a) during exercise at this intensity, possibly because of O(2) and/or metabolic limitations. (+info)
Lessons learned in applying the U.S. EPA proposed cancer guidelines to specific compounds.
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An expert panel was convened to evaluate the U.S. Environmental Protection Agency's "Proposed Guidelines for Carcinogen Risk Assessment" through their application to data sets for chloroform (CHCl3) and dichloroacetic acid (DCA). The panel also commented on perceived strengths and limitations encountered in applying the guidelines to these specific compounds. This latter aspect of the panel's activities is the focus of this perspective. The panel was very enthusiastic about the evolution of these proposed guidelines, which represent a major step forward from earlier EPA guidance on cancer-risk assessment. These new guidelines provide the latitude to consider diverse scientific data and allow considerable flexibility in dose-response assessments, depending on the chemical's mode of action. They serve as a very useful template for incorporating state-of-the-art science into carcinogen risk assessments. In addition, the new guidelines promote harmonization of methodologies for cancer- and noncancer-risk assessments. While new guidance on the qualitative decisions ensuing from the determination of mode of action is relatively straightforward, the description of the quantitative implementation of various risk-assessment options requires additional development. Specific areas needing clarification include: (1) the decision criteria for judging the adequacy of the weight of evidence for any particular mode of action; (2) the role of mode of action in guiding development of toxicokinetic, biologically based or case-specific models; (3) the manner in which mode of action and other technical considerations provide guidance on margin-of-exposure calculations; (4) the relative roles of the risk manager versus the risk assessor in evaluating the margin of exposure; and (5 ) the influence of mode of action in harmonizing cancer and noncancer risk assessment methodologies. These points are elaborated as recommendations for improvements to any revisions. In general, the incorporation of examples of quantitative assessments for specific chemicals would strengthen the guidelines. Clearly, any revisions should retain the emphasis present in these draft guidelines on flexibility in the use of scientific information with individual compounds, while simultaneously improving the description of the processes by which these mode-of-action data are organized and interpreted. (+info)
Marked differences between two isoforms of human pyruvate dehydrogenase kinase.
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Pyruvate dehydrogenase kinase (PDK) isoforms 2 and 3 were produced via co-expression with the chaperonins GroEL and GroES and purified with high specific activities in affinity tag-free forms. By using human components, we have evaluated how binding to the lipoyl domains of the dihydrolipoyl acetyltransferase (E2) produces the predominant changes in the rates of phosphorylation of the pyruvate dehydrogenase (E1) component by PDK2 and PDK3. E2 assembles as a 60-mer via its C-terminal domain and has mobile connections to an E1-binding domain and then two lipoyl domains, L2 and L1 at the N terminus. PDK3 was activated 17-fold by E2; the majority of this activation was facilitated by the free L2 domain (half-maximal activation at 3.3 microm L2). The direct activation of PDK3 by the L2 domain resulted in a 12.8-fold increase in k(cat) along with about a 2-fold decrease in the K(m) of PDK3 for E1. PDK3 was poorly inhibited by pyruvate or dichloroacetate (DCA). PDK3 activity was stimulated upon reductive acetylation of L1 and L2 when full activation of PDK3 by E2 was avoided (e.g. using free lipoyl domains or ADP-inhibited E2-activated PDK3). In marked contrast, PDK2 was not responsive to free lipoyl domains, but the E2-60-mer enhanced PDK2 activity by 10-fold. E2 activation of PDK2 resulted in a greatly enhanced sensitivity to inhibition by pyruvate or DCA; pyruvate was effective at significantly lower levels than DCA. E2-activated PDK2 activity was stimulated >/=3-fold by reductive acetylation of E2; stimulated PDK2 retained high sensitivity to inhibition by ADP and DCA. Thus, PDK3 is directly activated by the L2 domain, and fully activated PDK3 is relatively insensitive to feed-forward (pyruvate) and feed-back (acetylating) effectors. PDK2 was activated only by assembled E2, and this activated state beget high responsiveness to those effectors. (+info)
Effect of trichloroethylene and its metabolites, dichloroacetic acid and trichloroacetic acid, on the methylation and expression of c-Jun and c-Myc protooncogenes in mouse liver: prevention by methionine.
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Trichloroethylene (TCE), dichloroacetic acid (DCA), and trichloroacetic acid (TCA) are environmental contaminants that are carcinogenic in mouse liver. 5-Methylcytosine (5-MeC) in DNA is a mechanism that controls the transcription of mRNA, including the protooncogenes, c-jun and c-myc. We have previously reported that TCE decreased methylation of the c-jun and c-myc genes and increased the level of their mRNAs. Decreased methylation of the protooncogenes could be a result of a deficiency in S-adenosylmethionine (SAM), so that methionine, by increasing the level of SAM, would prevent hypomethylation of the genes. For 5 days, female B6C3F1 mice were administered, daily by oral gavage, either 1000 mg/kg body weight of TCE or 500 mg/kg DCA or TCA. At 30 min after each dose of carcinogen, the mice received, by ip injection, 0-, 30-, 100-, 300-, or 450-mg/kg methionine. Mice were euthanized at 100 min after the last dose of DCA, TCA, or TCE. Decreased methylation in the promoter regions of the c-jun and c-myc genes and increased levels of their mRNA and proteins were found in livers of mice exposed to TCE, DCA, and TCA. Methionine prevented both the decreased methylation and the increased levels of the mRNA and proteins of the two pro-tooncogenes. The prevention by methionine of DCA- TCA-, and TCE-induced DNA hypomethylation supports the hypothesis that these carcinogens act by depleting the availability of SAM. Hence, methionine would prevent DNA hypomethylation by maintaining the level of SAM. Furthermore, the results suggest that the dose of DCA, TCA, or TCE must be sufficient to decrease the level of SAM in order for these carcinogens to be active. (+info)