Evaluation of chlorinated benz[a]anthracene on hepatic toxicity in rats and mutagenic activity in Salmonella typhimurium.

Chlorinated benz[a]anthracenes (Cl-BaA) are halogenated aromatic compounds (typified by dioxins) found in the environment at relatively high concentrations. Fischer 344 rats were intragastrically administered 0, 1, or 10 mg of Cl-BaA or its parent compound benz[a]anthracene (BaA) per kg of body weight for 14 consecutive days. Both chemicals at 10 mg/kg/day inhibited the gain in body weight, and consequent increase in relative liver weight. Hepatic gene expression of cytochrome P450 (CYP) 1A1, 1A2, and 1B1 was significantly stimulated by administration of BaA (10 mg/kg/day) compared with the control. After administration of Cl-BaA, only the CYP1A2 gene was significantly induced, even at the lower dosage; CYP1A1 and 1B1 mRNA levels remained unchanged in Cl-BaA-treated rats compared with controls. To elucidate the role of such Cl-BaA exposure and induced CYPs at toxicity onset, we investigated the mutagenicity of BaA and Cl-BaA using Salmonella typhimurium TA98 and TA100. BaA and Cl-BaA at 10 µg/plate produced positive results in both strains in the presence of rat S-9. Incubation of Cl-BaA with recombinant rat CYP1A2 produced a significantly higher number of revertant colonies in TA98 and TA100 than in controls, but no such change was observed for BaA. In conclusion, BaA changes its own physiological and toxicological actions by its chlorination; (1) daily exposure to Cl-BaA selectively induces hepatic CYP1A2 in rats and (2) Cl-BaA induces frameshift mutations in the presence of CYP1A2, although BaA does not exert mutagenicity. This indicates that CYP1A2 may metabolize Cl-BaA to active forms.

Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter.

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis. Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2. Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.

The cytokine and chemokine profiles in rhabdomyolysis in a patient with Gaucher disease type II.

This report describes a patient with Gaucher disease type II who developed severe rhabdomyolysis. We treated him successfully and measured various cytokine and chemokine levels sequentially to elucidate the pathophysiology of rhabdomyolysis. The serum levels of interleukin-6, -8, -10, granulocyte colony-stimulating factor, interferon-gamma, and monocyte chemoattractant protein-1 were markedly elevated in the early phase of rhabdomyolysis. These findings indicate that cytokines and chemokines are related to the massive myolysis and regenerating process. A viral infection may have triggered rhabdomyolysis through exaggerated activation of macrophages in our patient. The profiles of cytokines and chemokines should be examined in further cases to increase our understanding of the pathophysiology of rhabdomyolysis.

Reduced carbohydrate intake in citrin-deficient subjects.

Citrin is the liver-type aspartate-glutamate carrier that resides within the inner mitochondrial membrane. Citrin deficiency (due to homozygous or compound heterozygous mutations in the gene SLC25A13) causes both adult-onset type II citrullinaemia (CTLN2) and neonatal intrahepatic cholestasis (NICCD). Clinically, CTLN2 is characterized by hyperammonaemia and citrullinaemia, whereas NICCD has a much more varied and transient presentation that can include multiple aminoacidaemias, hypoproteinaemia, galactosaemia, hypoglycaemia, and jaundice. Personal histories from CTLN2 patients have repeatedly described an aversion to carbohydrate-rich foods, and clinical observations of dietary and therapeutic outcomes have suggested that their unusual food preferences may be directly related to their pathophysiology. In the present study, we monitored the food intake of 18 Japanese citrin-deficient subjects whose ages ranged from 1 to 33 years, comparing them against published values for the general Japanese population. Our survey confirmed a marked decrease in carbohydrate intake, which accounts for a smaller proportion of carbohydrates contributing to the total energy intake (PFC ratio) as well as a shift towards a lower centile distribution for carbohydrate intake relative to age- and sex-matched controls. These results strongly support an avoidance of carbohydrate-rich foods by citrin-deficient patients that may lead to worsening of symptoms.

Clinical pictures of 75 patients with neonatal intrahepatic cholestasis caused by citrin deficiency (NICCD).

We clarified the clinical features of NICCD (neonatal intrahepatic cholestasis caused by citrin deficiency) by retrospective review of symptoms, management and long-term outcome of 75 patients. The data were generated from questionnaires to paediatricians in charge of the patients. Thirty of the patients were referred to hospitals before 1 month of age because of positive results in newborn screening (hypergalactosaemia, hypermethioninaemia, and hyperphenylalaninaemia). The other 45, the screen-negative patients, were referred to hospitals with suspected neonatal hepatitis or biliary atresia because of jaundice or discoloured stool. Most of the screen-negative patients presented before 4 months of age, and 11 had failure to thrive. Laboratory data showed elevated serum bile acid concentrations, hypoproteinaemia, low levels of vitamin K-dependent coagulation factors and hypergalactosaemia. Hypoglycaemia was detected in 18 patients. Serum amino acid analyses showed significant elevation of citrulline and methionine concentrations. Most of the patients were given a lactose-free and/or medium-chain triglyceride-enriched formula and fat-soluble vitamins. Symptoms resolved in all but two of the patients by 12 months of age. The two patients with unresolved symptoms suffered from progressive liver failure and underwent liver transplantation before their first birthday. Another patient developed citrullinaemia type II (CTLN2) at age 16 years. It is important to recognize that NICCD is not always a benign condition.

Molecular analysis of holocarboxylase synthetase deficiency: a missense mutation and a single base deletion are predominant in Japanese patients.

Holocarboxylase synthetase (HCS) deficiency is an inherited disease of biotin metabolism characterized by a unique pattern of organic aciduria, metabolic acidosis, and skin lesions. By analysis of five patients in four unrelated families, two mutations were identified: a transition from T to C which causes an amino-acid substitution of proline for leucine at position 237 (L237P) and a single deletion of guanine (delG1067) followed by premature termination. One patient was homozygous for the L237P mutation, three patients in two families were compound heterozygotes of the missense and deletion alleles, and the other patient was heterozygous for the L237P mutation. Inheritance was successfully demonstrated in all of the patients' families by a modified PCR followed by restriction enzyme digestion. The two mutations accounted for seven of eight mutant alleles, while neither mutation was detected in 108 normal healthy Japanese children (216 alleles). Transient expression in cultured fibroblasts from a patient showed that the L237P mutation was responsible for decreased HCS activity. These results suggest that the L237P and delG1067 mutations are frequent disease-causing mutations in Japanese patients with HCS deficiency. This PCR-based technique may therefore be useful for detecting mutations among Japanese patients.

A cluster of lysinuric protein intolerance (LPI) patients in a northern part of Iwate, Japan due to a founder effect. The Mass Screening Group.

Lysinuric protein intolerance is an autosomal recessive disease characterized by defective transport of the dibasic aminoacids. Mutational analysis of LPI patients in the northern part of Japan revealed that six were homozygous for the R410X mutation and two others were compound heterozygotes of R410X and other unknown mutations. In the population epidemiology study in a local cluster in the northern part of Iwate, ten heterozygotes were found in 1190 newborn babies leading to an estimated LPI incidence of 1/57,000. Polymorphism analysis revealed two major alleles, A and B, in intron 8. While the population frequency of allele A was 0.9 and that of allele B was 0.1 in the northern part of Japan the R410X mutations were exclusively on allele B in 31 chromosomes suggesting a founder effect. Genetic analysis in patients revealed strong linkage disequilibrium with D14S283 and TCRA indicating that the R410X mutation occurred before at least 130 generations ago (about 2600 years). The R410X mutation was shown to be useful as a molecular marker for screening LPI patients in the northern part of Japan.

Expression of proto-oncogenes in normal and tumor tissues of human skin.

The expression of c-fos, c-myc, Ha-ras, N-ras, EGF-receptor, and cardiac actin genes was examined in 7 normal epidermis, 3 cellular nevi, and 8 skin tumors including 6 malignant and 2 benign tumors of human origin. These genes were transcribed in most normal and tumor tissues, though no tumor-specific expression of proto-oncogenes (c-fos, c-myc, Ha-ras, and N-ras) could be detected. However, there was a characteristic parallelism between the expression of c-fos and c-myc in normal epidermis, while the parallelism was not always definite in skin tumors. The ratio of c-fos/c-myc transcripts in normal epidermis was constant compared with the expression of other genes examined. These data suggest that c-fos and c-myc are expressed in all normal skin tissues, and that maintenance of a constant ratio of c-fos/c-myc is closely related to ordered cell growth of the tissues.

Immunocytochemical localization of ornithine aminotransferase in rat ocular tissues.

Gyrate atrophy of the choroid and retina is a rare inherited form of chorioretinal degeneration due to a deficiency of ornithine aminotransferase (OAT). We localized the enzyme in rat ocular tissues using immunocytochemical procedures. Immunoreactivity was observed in the epithelia of ciliary body, iris, and lens. Retinal pigment epithelium and Müller cells were immunoreactive in the retina. A little immunoreactive product was found in the choroid. Our findings suggested that OAT plays an important role in ornithine metabolism in these ocular tissues.

Cryptomicrotia and short, stubby fingers with excess fingertip arch patterns in a mother and son.

We describe a malformation syndrome of bilateral cryptomicrotia, brachytelomesophalangy, hypoplastic toe nails, and excess fingertip arch patterns in a chromosomally and mentally normal mother and son. Familial occurrence of this malformation complex has not been described previously and the manifestations of the patients do not correspond to those of any known malformation syndromes. The disorder in this family may be attributable to the pleiotropic effect of an autosomal or an X-linked dominant gene.

Novel mutations of the glutaryl-CoA dehydrogenase gene in two Japanese patients with glutaric aciduria type I.

We identified three different point mutations in the glutaryl-CoA dehydrogenase (GCDH) gene in two unrelated Japanese patients with glutaric aciduria type I (GA-I). One patient was a homozygote for Arg355His and the other a compound heterozygote for Ser305Leu and Met339Val. Arg355His and Met339Val are mutations hitherto undescribed, and all three mutations are predicted to alter the secondary structure of GCDH. Molecular analysis is useful for definite diagnosis and/or prenatal diagnosis of GA-I.

Heterogeneous mutations in the glucose-6-phosphatase gene in Japanese patients with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by glucose-6-phosphatase (G6Pase) deficiency. It is characterized by short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. Various mutations have been reported in the G6Pase gene (G6PC). However, in Japanese patients, a g727t substitution was found to be the major cause of GSD-Ia, accounting for 20 of 22 mutant alleles [Kajihara et al., 1995], and no other mutations have been found in this population. We analyzed four Japanese GSD-Ia patients and identified three other mutations in addition to the g727t. They included two missense mutations (R83H and P257L) and one nonsense mutation (R170X). Each of the three mutations exhibited markedly decreased G6Pase activity when expressed in COS7 cells. A patient homozygous for R170X showed multiple episodes of profound hypoglycemia associated with convulsions, while P257L was associated with a mild clinical phenotype. The presence of R170X in three unrelated families may implicate that it is another important mutation in the etiology of GSD-Ia in Japanese patients. Thus, the detection of non-g727t mutations is also important in establishing the DNA-based diagnosis of GSD-Ia in this population.

Glycogen storage disease type Ia: molecular diagnosis of 51 Japanese patients and characterization of splicing mutations by analysis of ectopically transcribed mRNA from lymphoblastoid cells.

Glycogen storage disease type Ia (GSD-Ia) is an autosomal recessive disorder of glycogen metabolism caused by a deficiency of glucose-6-phosphatase (G6Pase) that is expressed in the liver, kidney, and intestinal mucosa. Clinical manifestations include short stature, hepatomegaly, hypoglycemia, hyperuricemia, and lactic acidemia. To elucidate a spectrum of the G6Pase gene mutations and their frequencies, we analyzed mutations in 51 unrelated Japanese patients with GSD-Ia. The most prevalent mutation was g727t, accounting for 88 of 102 mutant alleles examined, followed by R170X mutation, which accounted for 6 mutant alleles, and R83H mutation which was observed in 3 mutant alleles. In addition, 3 different, novel mutations, IVS1-1g

Influence of pimozide on hypothalamo-pituitary function in children with behavioral disorders.

Hypothalamo-pituitary functions were examined in thirteen children with behavioral disorders (six with hyperkinesia, four with autism, two with tic and one with schizophrenia) before and during treatment with pimozide, an antidopaminergic drug. The mean (+/- S.E.M.) basal serum PRL level (24.5 +/- 4.2 ng/ml) during pimozide treatment was significantly higher than that (12.4 +/- 3.2 ng/ml) before treatment. Hyperresponse of PRL to TSH releasing hormone (TRH) was observed in five (three with hyperkinesia, one with tic and one with autism) of the thirteen patients before treatment and in seven (four with hyperkinesia, two with autism and one with tic) during treatment. Mean TSH response during treatment was not significantly different from that before treatment. However, three of the four autistic children showed hyperresponse of TSH to TRH before treatment, whereas only one also showed a hyperresponse during treatment. The pimozide treatment had no demonstrable influence on GH or cortisol secretion in response to insulin-induced hypoglycemia, or on serum T4 and T3 levels.

Immunocytochemical localization of ornithine aminotransferase in human ocular tissues.

Gyrate atrophy of the choroid and retina is a rare inherited form chorioretinal degeneration caused by a deficiency of ornithine aminotransferase. We localized the enzyme in human ocular tissues using immunocytochemical procedures. Immunoreactivity was observed in the nonpigmented epithelium of the ciliary body and lens epithelium. In the retina, ganglion cells and some amacrine cells were immunoreactive. Pigmented granules made it difficult to identify immunoreactive products in the iris, pigmented epithelium of the ciliary body, choroid, and retinal pigment epithelium. Our findings suggested that ornithine aminotransferase plays an important role in ornithine metabolism in these oculartissues.

Crystallization and properties of human liver ornithine aminotransferase.

Ornithine aminotransferase [EC 2.6.1.13] was purified and crystallized from human liver by a procedure involving heat treatment, chromatographies on DEAE-cellulose, Octyl-Sepharose CL-4B and Sephadex G-200, and crystallization. The purified enzyme appeared to be homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated as 44,000 by sodium dodecyl sulfate electrophoresis and as 177,000 by sucrose density gradient centrifugation, indicating that the enzyme is tetrameric. Various properties of the enzyme from human liver are similar to those of the enzyme from rat liver, including its molecular weight, pH optimum, Km values for ornithine, alpha-ketoglutarate and pyridoxal phosphate and specificity for amino acceptor from ornithine. The amino acid compositions of the two enzymes also have certain similarities, but the enzymes differ in electrophoretic mobility and antigenicity: the human enzyme moved more slowly to the anode, and on immunodiffusion analysis, the single precipitin lines formed between anti-human enzyme serum or anti-rat liver enzyme and the enzyme from human liver or lymphoblastoid cells and the rat liver enzyme fused with spur formation.

Effects of individual mutations in the P-450(C21) pseudogene on the P-450(C21) activity and their distribution in the patient genomes of congenital steroid 21-hydroxylase deficiency.

Recent observations have suggested that the pathological mutations in human P-450(C21) deficiency are generated through gene conversion-like events between the functional gene [P-450(21)B] and the pseudogene [P-450(C21)A]. To address this point more extensively, we investigated the effects of the base changes in the A pseudogene on the P-450(21) activity by using the COS cell expression system. In addition to the defective mutations found previously in the pseudogene, four single base changes with amino acid substitutions of Pro(30), Ile(172), Val(282), or Arg(356) were further identified as causing complete [Arg(356)] or partial [Pro(30), Ile(172), and Val(282)] inactivation of P-450(C21). Blot hybridization analysis of patient DNAs using oligonucleotide probes specific for these mutations revealed that the splicing mutation in the 2nd intron was distributed most frequently in both simple-virilizing and salt-wasting forms. The mutation Ile(172) seemed to be frequent in patients with the less severe simple-virilizing form, whereas the mutation Arg(356), together with other most serious mutations reported previously, was preferentially associated with salt-wasting, the most severe form of the disease. In combination with the present results of the effects of various mutations on the P-50(C21) activity, a survey of the distribution of the various mutations in the patient genomes so far reported suggests that the heterogeneous clinical symptoms of this genetic disease are somehow related to the degree of attenuation of the activities of the mutated gene products.

Gyrate atrophy of the choroid and retina: decreased ornithine aminotransferase concentration in cultured skin fibroblasts from patients.

A sensitive and convenient radioisotopic assay for ornithine aminotransferase (OAT) and an enzyme immunoassay for human ornithine aminotransferase were developed for studying decrease in activity of this enzyme in gyrate atrophy of the choroid and retina with hyperornithinemia. Picogram amounts of human OAT could be detected by the enzyme immunoassay. The OAT activity in cultured fibroblasts from two patients was 6% and 2% of that in control fibroblasts, and did not increase on addition of a high concentration of vitamin B6. The decreased activity, assayed by enzyme immunoassay, was associated with decreased enzyme concentration, suggesting that deficiency of OAT in these patients is not due to production of a structurally altered enzyme lacking catalytic activity, but to decreased production of enzyme protein. When cells from patients were repeatedly subcultured, selective loss of both the activity and concentration of OAT was observed, without changes in activities of several other enzymes tested.

Detection of ornithine transcarbamylase deficiency heterozygotes by measuring of urinary uracil.

The importance of detecting heterozygosity for X-linked ornithine transcarbamylase deficiency is well known. Although the DNA analysis and the allopurinol loading tests are commonly used for this purpose, both methods require complicated procedures. In order to establish a simple test for detecting female heterozygotes, we examined the uracil and orotic acid in single-voided urine samples from 70 healthy women, and from 12 asymptomatic females with ornithine transcarbamylase deficiency. Based on the results of healthy women, we were able to determine a screening cut-off line of 11.9 micromol/mmol creatinine (mean +/- 1SD in logarithmic form) for uracil. Using this cut-off line, the sensitivity of OCT heterozygotes was 100%. We were also able to establish a second cut-off line of 28.9 micromol/mmol creatinine (mean +/- 3SD in logarithmic form) for diagnosis. Using this second cut-off line, the specificity of OCT heterozygotes was 100%. Our study has shown that the measurement of urinary uracil is a relatively simple and effective method for detecting female heterozygotes.

An undescribed subset of neonatal intrahepatic cholestasis associated with multiple hyperaminoacidemia.

Five patients of cholestatic jaundice and multiple hyperaminoacidemias were uncovered during neonatal mass screening for homocystinuria. All five patients had increased plasma levels of methionine, citrulline, tyrosine, threonine, phenylalanine, lysine and arginine. Compared with those of age-matched cholestatic disease controls, idiopathic neonatal hepatitis (n=9) and biliary atresia (n=14), plasma levels of three amino acids, citrulline, methionine, and threonine, were significantly greater, respectively (P<0.01). Liver biopsies examined in four patients uniformly showed diffuse hepatic fatty liver with micro- and macrovesicular droplets without giant cell transformation. Administration of fat-soluble vitamins and formula milk containing middle-chain triglyceride resulted in normalization of amino acid profiles by 6 weeks after the treatment. All liver function tests normalized by 17 months of age.