Kazuo Hidaka

The role of nitric oxide in paraquat-induced cytotoxicity in the human A549 lung carcinoma cell line

Paraquat (PQ) is a well-known pneumotoxicant that exerts its toxic effect by elevating intracellular levels of superoxide. In addition, production of pro-inflammatory cytokines has possibly been linked to PQ-induced inflammatory processes through reactive oxygen species (ROSs) and nitric oxide (NO). However, the role of NO in PQ-induced cell injury has been controversial. To explore this problem, we examined the effect of NO on A549 cells by exposing them to the exogenous NO donor NOC18 or to cytokines; tumor necrosis factor-α, interleukin-1 β and interferon-γ, as well as PQ. Although the exogenous NO donor on its own had no effect on the release of lactate dehydrogenase (LDH), remarkable release was observed when the cells were exposed to high concentrations of NOC18 and PQ. This cellular damage caused by 1 mM NOC18 plus 0.2 mM PQ was ascertained by phase contrast microscopy. On the other hand, NO derived from 25–50 μM NOC18 added into the medium improved the MTT reduction activity of mitochondria, suggesting a beneficial effect of NO on the cells. Incubation of A549 cells with cytokines increased in inducible NO synthase (iNOS) expression and nitrite accumulation, resulting in LDH release. PQ further potentiated this release. The increase in nitrite levels could be completely prevented by NOS inhibitors, while the leakage of LDH was not attenuated by the inhibition of NO production with them. On the other hand, ROS scavenging enzymes, superoxide dismutase and catalase, inhibited the leakage of LDH, whereas they had no effect on the increase in the nitrite level. These results indicate that superoxide, not NO, played a key role in the cellular damage caused by PQ/cytokines. Our in vitro models demonstrate that NO has both beneficial and deleterious actions, depending on the concentrations produced and model system used.

Identification of a point mutation associated with a silent phenotype of human serum butyrylcholinesterase – a case of familial cholinesterasemia

A point mutation which caused a silent phenotype of human serum butyrylcholinesterase (BChE) was identified in the DNA of a 47-year-old Japanese woman who visited our hospital complaining of hypertension. The propositus exhibited an unusually low level of BChE activity, whereas her younger sister and her daughter had intermediate levels of BChE activity and her elder sister a normal level. Immunologically, the amount of BChE protein in the serum of the propositus was normal. DNA sequence analysis of the propositus identified a point mutation at codon 199 (GCA --> GTA), resulting in a Ala --> Val substitution. This alteration is one downstream codon from the catalytic active site (Ser, 198). A family study showed her younger sister and her daughter to have the same mutation.

Gene analysis of genomic DNA from stored serum by polymerase chain reaction: identification of three missense mutations in patients with cholinesterasemia and ABO genotyping

We established a method to determine the butyrylcholinesterase genotype associated with a BCHE deficiency directly using multiple PCR from stored serum, which was stored at -70 degrees C for more than 30 years. PCR products from sera of six propositi were used for DNA sequence analysis. All of these BChE variants were characterized by a single nucleotide substitution. Four of them were homozygotes and demonstrated a C-->T single nucleotide point mutation at codon 100 from CCA (Pro) to TCA (Ser). The fifth case was a heterozygote of this mutation. The remaining one was a compound heterozygote showing a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and a G-->C transversion mutation at codon 365 from GGA (Gly) to CGA (Arg). Furthermore we developed a method to determine the ABO genotype from the same serum. These results indicated that serum is useful as a starting material for amplification of genomic DNA when fresh blood samples are not available.

Nonsense mutation in exon 2 of the butyrylcholinesterase gene: a case of familial cholinesterasemia

A point mutation that causes a silent phenotype for human serum butyrylcholinesterase (BChE) was proved by DNA analyses of a 64-year-old Japanese female who visited the hospital because of a common cold. The propositus and her two siblings showed extremely low BChE activity, but other family members (six individuals) manifested from intermediate to normal values of BChE activity. An immunological method revealed that the propositus and her two siblings showed absence of the BChE protein in serum. DNA sequence analysis of the propositus identified a point mutation at codon 400 (TGC-->TGA), resulting in the production of a stop codon. This alteration exists upstream of the Cys571 of the subunit, which forms a disulfide bridge with the Cys571 of another partner subunit.

Genetic analysis of a Japanese patient with butyrylcholinesterase deficiency

A patient (64‐year‐old, male) with familial cholinesterasemia caused by BChE deficiency was studied. DNA sequence analysis of all exons identified a point mutation, an A→G transition at codon 128, resulting in a Tyr→Cys substitution. The propositus showed extremely low BChE activity, but his other family members (three individuals) showed from intermediate to normal BChE activity. An immunological method revealed the absence of BChE protein in serum of the propositus. Both PCR primer introduced restriction analysis (PCR‐PIRA) and sequence analysis revealed all three family members to be heterozygotes for this mutation.

Changes in mRNAs of inducible nitric oxide synthase and interleukin-1β in the liver, kidney and lung tissues of rats acutely exposed to paraquat

Nitric oxide (NO) reacts with superoxide to form the potent oxidant peroxynitrite, which causes serious cell damage. Interleukin-1 beta (IL-1 beta) is known to be a strong activator of NO production via induction of inducible nitric oxide synthase (iNOS). Since paraquat (PQ) undergoes redox cycling in vivo, resulting in a constant generation of superoxide, peroxynitrite may be a pathogenetic factor in the oxidative cell damage. In this study, we have investigated whether mRNAs of iNOS and IL-1 beta are affected in rat liver, kidney and lung tissues by exposure to non-lethal and lethal doses of PQ. Suppression and then marked stimulation of the iNOS mRNA were observed in the liver tissues of rats exposed to a lethal dose of PQ, while the kidney and lung tissues showed little changes. We also detected nitrotyrosine in liver tissues of rats exposed to a lethal dose by immunohistochemistry, suggesting the simultaneous generation of NO and superoxide in liver injury during acute lethal PQ poisoning. On the other hand, the IL-1 beta mRNA in the liver tissues decreased throughout the experiments, suggesting that this cytokine is not responsible for stimulation of the iNOS gene. IL-1 beta mRNA in lung tissues in the non-lethal group showed an increase, with maximum levels at 16-24 h, while little changes were observed in iNOS mRNA in this organ. These data suggest that acute lethal poisoning and non-lethal poisoning by PQ undergo different mechanisms of action of NO and IL-1 beta systems; the former is due, at least in part, to an increase in NO production, while the latter is due to stimulation of IL-1 beta and/or other cytokines.

Water-restraint stress enhances methamphetamine-induced cardiotoxicity

Methamphetamine (MAP) and stress both cause a variety of cardiovascular problems. Stress also increases stimulant drug-seeking or drug-taking behavior by both humans and animals. In addition to the physiological effects on circulation, metabolism, and excretion, stress affects subjects responses to stimulant drugs such as MAP. However, the mechanisms underlying the drug-stress interactions remain unknown. In the present study, we assessed the effects of stress on myocardial responses to MAP in mice. Mice were injected with MAP (30mg/kg) immediately before exposure to water-restraint stress (WRS), which has often been used as a stressor in animal experiments. The combination of MAP with WRS produced a significant increase (p<0.01) in the leakage of proteins specific to myocardial damage and the levels of cytokines IL-6, TNF-α, and IL-10. The histological findings indicated the possibility that a combination of MAP with WRS induced cardiac myocytolysis. We also examined the expression of heat shock proteins (Hsps), which have cardioprotective effects. Administration of MAP alone significantly stimulated the RNA expressions of Hsp32, 60, 70, and 90 and the protein Hsp70 in cardiac muscles, whereas the expressions due to WRS or MAP plus WRS were not increased. These results reveal the fact that exposure to WRS depresses the induction of Hsps, in particular Hsp70, due to MAP injection, following to enhance MAP-induced myocardial damage. We believe that interactions between MAP and severe stress, including environmental temperature, affect the induction of Hsps, following to susceptibility of hosts to cardiotoxicity due to the stimulant drug.

First case of missense mutation (LDH-H:R171P) in exon 4 of the lactate dehydrogenase gene detected in a Japanese patient

AbstractComplete deficiency of lactate dehydrogenase (LDH) subunit H was identified in a 41-year-old woman with paralysis of her left lower limb. The propositus had extremely low LDH activity and five of her family members had levels of LDH activity that ranged from lower than normal to normal level. A transversion mutation at codon 171 (CGC→CCC), resulting in an Arg→Pro substitution was identified in her DNA sequence. A new NruI restriction site was introduced into the polymerase chain reaction (PCR) product by PCR-primer introduced restriction analysis (PCR-PIRA) using a specific mismatched primer. Digestion with NruI revealed that the propositus and her mother were, respectively, homozygous and heterozygous for this mutation.

Association between estrogen receptor α polymorphisms and equol production, and its relation to bone mass

To investigate the relationship between estrogen receptor polymorphisms and equol production and its effect on bone turnover, 139 workers (mean age 38.3+/-11.1 years) in Japan were recruited. Bone mineral density (BMD), bone turnover markers, and serum equol were measured at a health examination. DNA samples were prepared to detect the estrogen receptor alpha (ERalpha) polymorphism and were digested by PvuII. The number of equol producers was 57. No statistically significant differences were observed in bone mineral density and bone turnover markers between each ERalpha polymorphism and equol production. Since the adjusted odds ratio indicated that interaction itself decreased the risk of osteosono-assessment index (OSI) reduction using logistic regression analysis, further analysis was performed divided by each ERalpha polymorphism. Although the crude odds ratio showed no relationship between equol producers and non-producers, the adjusted odds ratio showed that equol producers with ERalpha pp had a significantly decreased risk of OSI reduction. Although this study was cross-sectional, both equol production and ERalpha polymorphism are closely associated with each other in relation to BMD.

Early differential gene expression of rat lung after exposure to paraquat

Paraquat (PQ), a quaternary nitrogen herbicide, is highly toxic to humans and animals. Acute poisoning and death due to PQ exposure have been reported over the past few decades. Excessive production of oxygen free radicals has been proposed to play an important role in the pulmonary pathology. The aim of the present work was to evaluate the implications for genes that are regulated by oxidative stress at the early stage of PQ exposure in rat lungs. We performed differential display RT-PCR (DD-PCR) on total RNA extracted from rat lungs after injection of 20mg per kg body weight. The experimental DD-PCR conditions, primer length and annealing temperature, were adjusted to improve reproducibility, and 19 differentiated clones were isolated. Sequence analysis followed by conventional RT-PCR and real-time RT-PCR analyses were used to confirm the results. Four clones were finally determined to be significantly affected. These genes were mRNAs for plasma phospholipid transfer protein (PLTP), CL1BA protein, (latrophilin: LPH), and alphaII-spectrin as well as one unknown gene. We demonstrated the distribution of mRNA expression of one gene, LPH, in lung tissues. The present study suggests that 20mg per kg intraperitoneal PQ affects the expression of numerous genes in the lung at 3 h, the onset of pulmonary injury, and that the four genes specified may be major contributors to serious lung injury due to PQ exposure.