Molecular identification of mutations associated with anti-tuberculosis drug resistance among strains of Mycobacterium tuberculosis.

BACKGROUND: Understanding the etiologic organism, antimicrobial resistance mechanisms, and transmission of multidrug-resistant tuberculosis (MDR-TB) can be of great value in optimizing strategies to control and prevent its development and transmission. METHODS: One hundred and fifty-five Mycobacterium tuberculosis complex isolates from patients with pulmonary tuberculosis (TB) in Cairo, Egypt were studied. In vitro drug susceptibility testing against rifampin (RIF), isoniazid (INH), streptomycin (SM), ethambutol (EMB), and pyrazinamide (PZA) was performed. Resistance was studied by the standard agar proportion method. Single strand conformation polymorphism (SSCP) and DNA sequence analysis were used to detect mutations in the genes that encode resistance to rpoB, katG, rpsL, and embB. RESULTS: Among 155 consecutive M. tuberculosis isolates, 25 (16.1%) were MDR-TB; 13 of these were from newly diagnosed untreated cases, 12 were from re-treated cases, and none of the MDR-TB isolates had matching IS6110 fingerprints. Among the MDR-TB isolates, rpoB mutations were found in 76% of RIF-resistant isolates, katG mutations were found in 47.1% of INH-resistant isolates, rpsL mutations were found in 55.6% of SM-resistant isolates, and embB mutations were found in 36.4% of EMB-resistant isolates. CONCLUSIONS: No major differences were found in the frequencies of mutations or types of amino acid substitution between newly diagnosed untreated cases and re-treated cases. The high prevalence of MDR-TB at this hospital underscores the need for continuous monitoring of strains and antimicrobial resistance.

Characterization of IS6110 restriction fragment length polymorphism patterns and mechanisms of antimicrobial resistance for multidrug-resistant isolates of Mycobacterium tuberculosis from a major reference hospital in Assiut, Egypt.

We evaluated 25 Mycobacterium tuberculosis isolates from patients at a major Egyptian reference hospital in Assiut, Egypt, who had been treated for at least 1 year for tuberculosis. Typing patterns (IS6110) were diverse, and multidrug resistance was found among 11 (44%) of the isolates. Mutations associated with antimicrobial drug resistance were found in rpoB, katG, rpsL, and embB in the resistant isolates.

Overexpression of glutamine: fructose-6-phosphate amidotransferase in the liver of transgenic mice results in enhanced glycogen storage, hyperlipidemia, obesity, and impaired glucose tolerance.

To examine the effect of increased hexosamine flux in liver, the rate-limiting enzyme in hexosamine biosynthesis (glutamine:fructose-6-phosphate amidotransferase [GFA]) was overexpressed in transgenic mice using the PEPCK promoter. Liver from random-fed transgenic mice had 1.6-fold higher GFA activity compared with nontransgenic control littermates (276 +/- 24 pmol x mg(-1) x min(-1) in transgenic mice vs. 176 +/- 18 pmol x mg(-1) x min(-1) in controls, P < 0.05) and higher levels of the hexosamine end product UDP-N-acetyl glucosamine (288 +/- 11 pmol/g in transgenic mice vs. 233 +/- 10 pmol/g in controls, P < 0.001). Younger transgenic mice compared with control mice had lower fasting serum glucose (4.8 +/- 0.5 mmol/l in transgenic mice vs. 6.5 +/- 0.8 mmol/l in controls, P < 0.05) without higher insulin levels (48.0 +/- 7.8 pmol/l in transgenic mice vs. 56.4 +/- 5.4 pmol/l in controls, P = NS); insulin levels were significantly lower in transgenic males (P < 0.05). At 6 months of age, transgenic animals had normal insulin sensitivity by the hyperinsulinemic clamp technique. Hepatic glycogen content was higher in the transgenic mice (108.6 +/- 5.2 pmol/g in transgenic mice vs. 32.8 +/- 1.3 micromol/g in controls, P < 0.01), associated with an inappropriate activation of glycogen synthase. Serum levels of free fatty acids (FFAs) and triglycerides were also elevated (FFAs, 0.67 +/- 0.03 mmol/l in transgenic mice vs. 0.14 +/- 0.01 in controls; triglycerides, 1.34 +/- 0.15 mmol/l in transgenic mice vs. 0.38 +/- 0.01 in controls, P < 0.01). Older transgenic mice became heavier than control mice and exhibited relative glucose intolerance and insulin resistance. The glucose disposal rate at 8 months of age was 154 +/- 5 mg x kg(-1) x min(-1) in transgenic mice vs. 191 +/- 6 mg x kg(-1) x min(-1) in controls (P < 0.05). We conclude that hexosamines are mediators of glucose sensing for the regulation of hepatic glycogen and lipid metabolism. Increased hexosamine flux in the liver signals a shift toward fuel storage, resulting ultimately in obesity and insulin resistance.

Hexosamines regulate leptin production in human subcutaneous adipocytes.

The hexosamine biosynthetic pathway has recently been proposed as a mechanism through which cells "sense" nutrient flux to regulate leptin release. This study was undertaken to examine the regulation of leptin production by hexosamines in human adipocytes. Adipose tissue UDP-N-acetylglucosamine, an end product of hexosamine biosynthesis, was elevated 3.2-fold, and ob messenger ribonucleic acid was elevated 2-fold in the sc adipose tissue of 17 obese [body mass index (BMI), 41.3+/-12.0 kg/m2; age, 31+/-5 yr] subjects compared to 14 lean (BMI, 23.4+/-1.6 kg/m2; age, 33+/-11 yr) subjects. Serum leptin was increased 2.7-fold in the obese subjects. A significant positive relationship was found between adipose tissue UDP-N-acetylglucosamine and BMI (Spearman correlation = 0.576; P = 0.0007) and between UDP-N-acetylglucosamine and serum leptin (Spearman correlation = 0.4650; P = 0.0145). Treatment of isolated sc adipocytes with 1 mmol/L glucosamine, an intermediate product in UDP-N-acetylglucosamine biosynthesis, increased leptin release 21.4+/-17.6% (mean +/- SD) over control (P = 0.0365) and 74.5+/-82.8% over control (P = 0.0271) in adipocytes from lean (BMI, 23.2+/-1.6 kg/m2; n = 6) and obese (BMI, 55.4+/-13.0 kg/m2,; n = 9) subjects, respectively, by 48 h of culture. Inhibition of UDP-N-acetylglucosamine biosynthesis with 6-diazo-5-oxo-norleucine reduced glucose-stimulated leptin release from cultured adipocytes 21.8+/-32.4% (P = 0.0395; n = 12) and ob gene expression 19.9+/-18.9% (P = 0.0208; n = 8) by 48 h of treatment. These findings suggest that hexosamine biosynthesis regulates leptin production in human adipose tissue.

Transgenic mice with increased hexosamine flux specifically targeted to beta-cells exhibit hyperinsulinemia and peripheral insulin resistance.

Hexosamines have been shown to mediate effects of hyperglycemia and so-called "glucose toxicity" in insulin-sensitive tissues. To determine the effects of hexosamines on insulin synthesis and secretion, transgenic mice were created to overexpress the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), specifically in beta-cells. GFA activity in islets of heterozygous transgenic mice was elevated 76% compared with littermate controls. The increased GFA activity led to 1.4- and 2.1-fold increased pancreatic insulin content in 2- and 10-month-old transgenic mice, respectively (P < 0.005). Fasting insulin levels were 1.6-fold higher than in littermate controls (P < 0.05). Hyperinsulinemia was evident despite a 28% reduction in insulin mRNA levels. The fasting glucose levels in the transgenic mice equaled that of controls aged 2-4 months but exceeded that of the controls aged 6-10 months (means +/- SE 6.9 +/- 0.2 vs. 5.9 +/- 0.2 mmol/l, P < 0.001). By 8 months, the males were overweight and mildly diabetic (fasting glucose 8.8 +/- 0.5 mmol/l) despite persistent hyperinsulinemia. Insulin resistance was confirmed in both males and females using the euglycemic-hyperinsulinemic clamp technique; glucose disposal rates decreased by 48% in transgenic mice (P < 0.01). Triglyceride levels did not differ, and free fatty acid levels were lower in the transgenic animals. ATP levels were unchanged in the transgenic islets. We conclude that hexosamine biosynthesis is involved in the regulation of insulin content in beta-cells by glucose. Increased hexosamine flux in the beta-cell results in hyperinsulinemia, insulin resistance, and (in males) mild type 2 diabetes.

Phenotypic characterization of pncA mutants of Mycobacterium tuberculosis.

We examined the correlation of mutations in the pyrazinamidase (PZase) gene (pncA) with the pyrazinamide (PZA) resistance phenotype with 60 Mycobacterium tuberculosis isolates. PZase activity was determined by the method of Wayne (L. G. Wayne, Am. Rev. Respir. Dis. 109:147-151, 1974), and the entire pncA nucleotide sequence, including the 74 bp upstream of the start codon, was determined. PZA susceptibility testing was performed by the method of proportions on modified Middlebrook and Cohn 7H10 medium. The PZA MICs were > or =100 microg/ml for 37 isolates, 34 of which had alterations in the pncA gene. These mutations included missense substitutions for 24 isolates, nonsense substitutions for 3 isolates, frameshifts by deletion for 4 isolates, a three-codon insertion for 1 isolate, and putative regulatory mutations for 2 isolates. Among 21 isolates for which PZA MICs were <100 microg/ml, 3 had the same mutation (Thr47-->Ala) and 18 had the wild-type sequence. For the three Thr47-->Ala mutants PZA MICs were 12.5 microg/ml by the method of proportions on 7H10 agar; two of these were resistant to 100 microg of PZA per ml and the third was resistant to 800 microg of PZA per ml by the BACTEC method. In all, 30 different pncA mutations were found among the 37 pncA mutants. No PZase activity was detected in 35 of 37 strains that were resistant to > or =100 microg of PZA per ml or in 34 of 37 pncA mutants. Reduced PZase activity was found in the three mutants with the Thr47-->Ala mutation. This study demonstrates that mutations in the pncA gene may serve as a reliable indicator of resistance to > or =100 microg of PZA per ml.

Hexosamines stimulate leptin production in transgenic mice.

Hexosamine flux has been shown to mediate aspects of nutrient sensing in insulin sensitive tissues and has been hypothesized to represent a satiety signal that results in shunting of fuel toward storage as fat. It has been recently reported that in vitro treatment of fat and muscle cells with hexosamines and acute glucosamine infusion in intact rats stimulate leptin secretion. In order to investigate the effects of chronic, physiologic increases in hexosamine flux on leptin we have examined leptin mRNA and serum leptin in mice overexpressing the rate-limiting enzyme for hexosamine synthesis, GFA, in muscle and fat. Increased levels of UDP-N-acetylglucosamine, the principal end-product of the hexosamine pathway were seen in transgenic fat, consistent with the overexpression of GFA. After overnight fasting, the transgenic mice were hyperleptinemic compared to littermate controls (4.5+/-0.5 ng/ml in transgenic, 2.8+/-0.2 in control, p = 0.005) despite equal body weights. In the random-fed state, the leptin levels of control mice increased to 4.1+/-0.5 ng/ml (p = 0.01) whereas the leptin levels in the transgenics did not increase any further (3.7+/-0.4 ng/ml). Leptin mRNA levels were also increased in transgenic fat (2.7+/-0.6 in transgenic compared to 0.8+/-0.2 in control, arbitrary units normalized to actin, p < 0.007). Despite increased leptin, the transgenic animals did not have lower body fat content. We conclude that hexosamine flux in fat regulates leptin synthesis and secretion.

Evaluation of the invader assay, a linear signal amplification method, for identification of mutations associated with resistance to rifampin and isoniazid in Mycobacterium tuberculosis.

We evaluated a recently described linear signal amplification method for sensitivity and specificity in detecting mutations associated with resistance to rifampin (RIF) and isoniazid (INH) in Mycobacterium tuberculosis. The assay utilizes the thermostable flap endonuclease Cleavase VIII, derived from Archaeoglobus fulgidus, which cleaves a structure formed by the hybridization of two overlapping oligonucleotide probes to a target nucleic acid strand. This method, termed the Invader assay, can discriminate single-base differences. Nine pairs of probes, encompassing five mutations in rpoB and katG that are associated with resistance to either RIF or INH, as well as the corresponding wild-type (drug-susceptible) alleles, were tested using amplified DNA. Fluorescent-labeled cleavage products, ranging from 4 to 13 nucleotides in length, depending on the genotype of the test sample, were separated by denaturing polyacrylamide (20 to 24%) gel electrophoresis and then detected by scanning. All nine alleles could be identified and differentiated on the basis of product size. Multiple mutations at a specific rpoB nucleotide in target PCR products could be identified, as could mutants that were present at > or =0.5% of the total population of target sequences. The Invader assay is a sensitive screen for some mutations associated with antituberculosis drug resistance in amplified gene regions.

Identification of Mycobacterium species by PCR-restriction fragment length polymorphism analyses using fluorescence capillary electrophoresis.

We developed a scheme for the rapid identification of Mycobacterium species based upon PCR amplification of polymorphic genetic regions with fluorescent primers followed by restriction and analysis by fluorescence capillary electrophoresis. Mycobacterium species were identified by restriction enzyme analysis of a 439-bp segment of the 65-kDa heat shock protein gene (labeled [both strands] at the 5' end with 4,7,2',7'-tetrachloro-6-carboxyfluorescein) using HaeIII and BstEII and of a 475-bp hypervariable region of the 16S rRNA gene (labeled [both strands] at the 5' end with 6-carboxyfluorescein) using HaeIII and CfoI. Samples were analyzed on an automated fluorescence capillary electrophoresis instrument, and labeled fragments were sized by comparison with an internal standard. DNA templates were prepared with pure cultures of type strains. In all, we analyzed 180 strains, representing 22 Mycobacterium species, and obtained distinctive restriction fragment length polymorphism (RFLP) patterns for 19 species. Three members of the Mycobacterium tuberculosis complex had a common RFLP pattern. A computerized algorithm which eliminates subjectivity from pattern interpretation and which is capable of identifying the species within a sample was developed. The convenience and short preparatory time of this assay make it comparable to conventional methodologies such as high-performance liquid chromatography and hybridization assays for identification of mycobacteria.

Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione.

Hexosamines have been hypothesized to mediate aspects of glucose sensing and toxic effects of hyperglycemia. For example, insulin resistance results when the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), is overexpressed in muscle and adipose tissue of transgenic mice. The glucose infusion rates required to maintain euglycemia at insulin infusion rates of 0.5, 2, 15, and 20 mU/kg x min were 39-90% lower in such transgenic mice, compared with their control littermates (P < or = 0.01). No differences were observed in hepatic glucose output, serum insulin levels, or muscle ATP levels. Uptake of 2-deoxyglucose, measured under conditions of hyperinsulinemia, was significantly lower in transgenic hindlimb muscle, compared with controls (85.9 +/- 17.8 vs. 166.8 +/- 15.1 pmol deoxyglucose/g x min). The decrease in glucose uptake by transgenic muscle was associated with a disruption in the translocation of the insulin-stimulated glucose transporter GLUT4. Fractionation of muscle membranes on a discontinuous sucrose gradient revealed that insulin stimulation of control muscle led to a 28.8% increase in GLUT4 content in the 25% fraction and a 61.2% decrease in the 35% fraction. In transgenic muscle, the insulin-stimulated shifts in GLUT4 distribution were inhibited by over 70%. Treatment of the transgenic animals with the thiazolidinedione troglitazone completely reversed the defect in glucose disposal without changing GFA activity or the levels of uridine 5'-diphosphate-N-acetylglucosamine. Overexpression of GFA in skeletal muscle thus leads to defects in glucose transport similar to those seen in type 2 diabetes. These data support the hypothesis that excess glucose metabolism through the hexosamine pathway may be responsible for the diminished insulin sensitivity and defective glucose uptake that are seen with hyperglycemia.

Evaluation of a line probe assay kit for characterization of rpoB mutations in rifampin-resistant Mycobacterium tuberculosis isolates from New York City.

A commercial line probe assay kit (Inno-LiPA Rif.TB) for rapid identification of mutations in the rpoB gene associated with rifampin resistance in Mycobacterium tuberculosis was evaluated with a collection of 51 rifampin-resistant strains. Nine distinct rpoB mutations were identified. Concordances with automated sequence results for five wild-type kit probes and four probes for specific mutations were 94.1 and 100%, respectively. Overall concordance of the line probe assay kit with phenotypic rifampin susceptibility testing results was 90.2%.

Characterization of streptomycin resistance mechanisms among Mycobacterium tuberculosis isolates from patients in New York City.

From a collection of 367 isolates of Mycobacterium tuberculosis from patients in New York City in 1994, 45 isolates (12.3%) were resistant in vitro to 2 micrograms or more of streptomycin (SM) per ml. We further evaluated these isolates for levels of SM resistance and for mutations previously associated with resistance in the rpsL (S12 ribosomal protein) gene and the rrs (16S rRNA)-coding region. Twenty-four isolates, representing nine distinct patterns of susceptibility to antituberculosis drugs, were resistant to 500 micrograms of SM per ml and shared a common point mutation at nucleotide 128 in the rpsL gene. This mutation, which substitutes lysine for arginine in the S12 ribosomal binding protein, was not present in isolates with low-level SM resistance or in SM-susceptible control isolates. Among 20 isolates with low-level SM resistance, one possessed a substitution (C-->G865) in the 912 loop of the rrs gene. No mutations in the 530 loop of the rrs coding region were detected, suggesting the presence of an alternative SM resistance mechanism in 19 isolates. Single-strand conformation polymorphisms of mutants were readily detected by a nonradioactive gel screen.

Antithyroid effects in vivo and in vitro of vitexin: a C-glucosylflavone in millet.

Millet diets rich in C-glycosylflavones (C-GF) are goitrogenic, and its three most abundant C-GF inhibit in vitro thyroid peroxidase, suggesting that these compounds are the goitrogens in millet. However, proof of a cause and effect relationship between C-GF and goitrogenesis requires a demonstration of in vivo antithyroid activity by the purified isolated compounds. Vitexin, one of the three major C-GF in millet, was used to test this hypothesis. Twenty-four female Wistar rats, divided into groups of six rats each and fed Purina iodine-rich diet (12 micrograms I-/day.rat), were administered acutely by gastrointestinal tube goitrogen-free water (controls), methimazole (0.5 mumol), and vitexin (20 and 80 mumol). 125I (1 microCi) was injected ip 1 h later, and the rats were killed 2 h after the injection. The thyroid glands were removed and analyzed for their content of total 125I and 125I-labeled compounds. Rats given vitexin, in contrast to those receiving methimazole, did not show suppressed thyroid 125I uptake. However, significant inhibition of the coupling mechanism (high 125I-labeled monoiodotyrosine plus diiodotyrosine/125T3 plus T4 ratio and low 125T3 and T4 concentrations) did occur with the highest dose of vitexin. These results provide direct evidence in vivo of C-GF antithyroid activity, strongly supporting the concept that C-GF are the goitrogens in millet.

Bioluminescence method to evaluate antimicrobial agents against Mycobacterium avium.

Plasmid pLUC10, carrying the firefly luciferase gene, was transformed by electroporation into Mycobacterium avium A5. Bioluminescence production by strain A5(pLUC10), as measured in a microdilution plate luminometer, was approximately 1 relative light unit per 2 x 10(6) viable bacilli, whereas it was 0.0005 relative light unit for an equal number of parental cells. The susceptibility of strain A5(pLUC10) to eight concentrations of each of eight antimicrobial agents was evaluated by the luciferase microplate assay in parallel with a conventional broth macrodilution method with antimicrobial agents. Decreases in bioluminescence to levels that were < or = 10% of those of drug-free controls were observed in microplate wells containing inhibitory concentrations of drugs in as few as 3 days. The close correlation of these inhibitory concentrations with the MICs determined by a conventional broth macrodilution method suggests that the luciferase microplate method may offer a convenient and reliable means of evaluating the in vitro activities of antimicrobial agents against the M. avium complex.

Antithyroid effects in vivo and in vitro of babassu and mandioca: a staple food in goiter areas of Brazil.

Babassu (Orbignya phalerata), a palm-tree coconut fruit, mixed with mandioca (Manihot utilissima) is the staple food of people living in the endemic goiter area of Maranhao in Brazil, where goiter prevalence among schoolchildren was still 38% in 1986 despite an adequate iodine intake in most of the population. Therefore, the question arose as to whether or not the ingestion of babassu alone or mixed with mandioca contributed to the persistence of endemic goiter in this area of Brazil. In this investigation we examined the potential antithyroid effects of babassu and mandioca by means of in vivo studies in Sprague-Dawley rats, in vitro studies in porcine thyroid slices and using a purified porcine thyroid peroxidase (TPO) system. Samples of various edible parts of babassu and mandioca flour were homogenized and extracted in goitrogen-free water (GFW) for in vivo experiments, and in methanol (100 g/l), GFW or 0.06 mol/l phosphate buffer (pH 7.0) for in vitro experiments. The edible parts of babassu produced significant in vivo antithyroid effects (p < 0.05- < 0.001) in rats on a high iodine intake (14 micrograms I- day-1.rat-1), as well as distinct and reproducible antithyroid and anti-TPO activities in both in vitro systems, their action being similar to that of the thionamide-like antithyroid drugs propylthiouracil and methimazole.(ABSTRACT TRUNCATED AT 250 WORDS)

Detection of the 10-kD antigen structural gene in mycobacteria by using the polymerase chain reaction.

The structural gene encoding the 10-kD antigen from Mycobacterium tuberculosis was amplified by the polymerase chain reaction. The 297-base-pair (bp) product was detected among 45 strains representing 14 mycobacterial species, but was absent from 11 species related to the mycobacteria. The gene was localized to a approximately 2000-bp SstII restriction fragment of the organisms' chromosomes.

Characterization of glycopeptide-resistant enterococci from U.S. hospitals.

We examined 105 clinical isolates of glycopeptide-resistant enterococci collected from 31 U.S. hospitals in 14 states during May 1988 to July 1992. The isolates included 82 Enterococcus faecium, 8 E. faecalis, 6 Enterococcus spp., 5 E. gallinarum, 3 E. casseliflavus, and 1 E. raffinosus. The isolates were categorized into the following four phenotypes of glycopeptide resistance on the basis of their MIC patterns: (i) 70 VanA (vancomycin [Vm] MIC, > or = 64 micrograms/ml; teicoplanin [Tei] MIC, 16 to > or = 128 micrograms/ml), (ii) 26 VanB (Vm MIC, 16 to 1,024 micrograms/ml; Tei MIC, < or = 2 micrograms/ml), (iii) 5 VanC (Vm MIC, 4 to 16 micrograms/ml; Tei MIC, < or = 2 micrograms/ml) in E. gallinarum, and (iv) 3 E. casseliflavus and 1 E. raffinosus isolates for which Vm MICs were 4 to 16 micrograms/ml and Tei MICs were < or = 1 micrograms/ml were called unclassified. Of the 101 isolates with the VanA, VanB, and VanC phenotypes, 99 were confirmed by production of a specific 1,030-, 433-, or 796-bp polymerase chain reaction product, respectively, and hybridization with the respective gene probe. The vanA gene was also detected in the E. raffinosus isolate for which the Vm MIC was 16 micrograms/ml and the Tei MIC was 1 microgram/ml. The vanA gene was located on either a 34- or a 60-kb plasmid in all of the U.S. isolates examined. Pulsed-field gel electrophoresis demonstrated both intrahospital and interhospital diversity among Vmr enterococci in the United States and was more useful than plasmid analysis for epidemiologic studies.

A rapid method for screening antimicrobial agents for activities against a strain of Mycobacterium tuberculosis expressing firefly luciferase.

We developed a rapid method to screen the efficacy of antimicrobial agents against Mycobacterium tuberculosis. A restriction fragment carrying a promoterless firefly luciferase gene was cloned into a 4,488-bp shuttle vector, pMV261, and luciferase was expressed under the control of a mycobacterial heat shock promoter. The resulting plasmid, pLUC10, was introduced by electroporation into the avirulent strain M. tuberculosis H37Ra. Luciferase assays of sonic lysates of Triton X-100-treated cells of M. tuberculosis H37Ra(pLUC10) yielded bioluminescence in excess of 1,000 relative light units/approximately 10(9) tubercle bacilli, compared with 0.0025 for the same number of parental cells. A 48-h microdilution antimicrobial agent-screening assay using this strain was developed.

Antithyroid and goitrogenic effects of coal-water extracts from iodine-sufficient goiter areas.

Goiter in iodine-sufficient areas has been linked to water-borne goitrogens in watersheds and aquifers rich in coal and shale. In the present study, the potential antithyroid and goitrogenic effects of coal-water extracts (CWE) were investigated in vivo in rats after chronic and acute oral administration of CWE, and in vitro by a thyroid peroxidase (TPO) enzyme system. CWE was prepared by continuous extraction of ground (40 mesh) Appalachian coal with goitrogen-free water (GFW). Female Buffalo rats fed on Purina iodine-rich diet (12 micrograms I-/day/rat), were given ad lib CWE (50 mg/ml; approximately 20 mL/day/rat) or GFW (controls) for 2 months. At the end of the experiment, 125I 1 microCi, was injected i.p. and 4 h later the thyroid glands were removed, weighed, and analyzed histologically and for total 125I and 125I-labeled compounds. Rats on CWE had larger thyroid glands [7.2 +/- 0.3 mg/100 g (mean +/- SE) vs 5.0 +/- 0.5 controls; p < 0.005] with distinct histological changes of smaller thyroid follicles, some with columnar epithelium, and with more dense colloid than in controls, and had significant inhibition of the coupling mechanism for production of thyroid hormones [125MIT + DIT/125T3 + T4: 5.1 +/- 0.2 vs 3.9 +/- 0.1 controls, p < 0.005; and 125T3 + T4 (%): 10.6 +/- 0.3 vs 12.6 +/- 0.4 controls, p < 0.005]. Female Sprague-Dawley rats under the same conditions as Buffalo rats were given acutely by GI tube 2 mL of CWE (5 g/mL) or GFW (controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Antithyroid effects of coal-derived pollutants.

Endemic goiter in iodide-sufficient areas of the United States and Colombia has been linked to watersheds rich in coal and shale, which several reports suggest are the source of water-borne goitrogens. In this report the potential antithyroid activities of aqueous coal and shale extracts and of compounds identified in aqueous effluents from coal conversion processes were assayed in thyroid peroxidase (TPO) and thyroid slice systems. Aqueous extracts of coal and black shale were potent inhibitors of TPO or 125I organification by thyroid slices. The most abundant water-soluble compounds derived from coal are dihydroxy-phenols, thiocyanate, disulfides, and hydroxypyridines. The dihydroxyphenols resorcinol, 2-methylresorcinol, and 5-methylresorcinol (orcinol) were 26.7, 22.5, and 7.2 times more potent, respectively, than the antithyroid drug 6-propylthiouracil (PTU). Other dihydroxyphenols and thiocyanate were less potent but comparable in activity to PTU. All dihydroxypyridines and 3-hydroxypyridine produced inhibitory effects comparable to PTU. None of the disulfides inhibited TPO. The antiperoxidase effects of combinations of two dihydroxyphenols or one dihydroxyphenol and SCN were additive, whereas the effects of a combination of four dihydroxyphenols at threshold inhibitory concentrations were synergistic, resulting in net effects equivalent to or greater than the sum of the individual effects. Thus, antithyroid effects may be greatly amplified by exposure to multiple coal-derived goitrogens and could be many times that produced by any one of the contributing pollutants. These results demonstrate that potent water-borne goitrogens are derived from coal and shale and that their contamination of water supplies could pose a serious threat of thyroid disorders.