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Biomedical subjects

Tomoyuki Kawada

Publications and source records attributed to Tomoyuki Kawada.

At least 19 recordsLinked to original sources
PubMed2006

Healthy lifestyles are associated with higher levels of perforin, granulysin and granzymes A/B-expressing cells in peripheral blood lymphocytes.

OBJECTIVE: It is well documented that natural killer (NK) cells provide host defense against tumors and viruses. We previously showed that lifestyle affects human NK and LAK activities. In order to explore the underlying mechanism, we investigated the effect of lifestyle on intracellular perforin, granulysin, and granzymes A/B in peripheral blood lymphocytes (PBL). METHODS: 114 healthy male subjects, aged 20-59 years, from a large company in Osaka, Japan were selected with informed consent. The subjects were divided into groups reporting good, moderate, and poor lifestyles according to their responses on a questionnaire regarding eight health practices (cigarette smoking, alcohol consumption, sleeping hours, working hours, physical exercise, eating breakfast, balanced nutrition, and mental stress). Peripheral blood was taken, and numbers of NK, T, perforin, granulysin, and granzymes A/B-expressing cells in PBL were measured by flow cytometry. RESULTS: Subjects with good or moderate lifestyle showed significantly higher numbers of NK, and perforin, granulysin, and granzymes A/B-expressing cells and a significantly lower number of T cells in PBL than subjects with poor lifestyle. Among the eight health practices, cigarette smoking, physical exercise, eating breakfast, and balanced nutrition significantly affect the numbers of NK, T cells, perforin, granulysin, and/or granzymes A/B-expressing cells, and alcohol consumption significantly affects the number of granzyme A-expressing cells. On the other hand, mental stress, sleeping, and working hours had no effect on those parameters. CONCLUSIONS: Taken together, these findings indicate that poor lifestyle significantly decreases the numbers of NK, perforin, granulysin, and granzymes A/B-expressing cells in PBL.

Adult↗
PubMed2006

Actigraphic predictors of the depressive state in students with no psychiatric disorders.

BACKGROUND: Insomnia is one of the characteristics of the early depressive state. One-third of university students has a mild depressive state. In this study, we examined whether the depressive state would be correlated with the actigraphic-derived daytime activity levels (DAL) and total sleep time (TST) in students with no psychiatric disorders. METHOD: Consenting volunteers were monitored at home for 3-4 days with a wrist actigraph. The DALs for 3 h before noon (DAL-M) and after 13:00 h (DAL-A) were calculated. The states of sleep and wakefulness were judged based on the activity levels, using a special algorithm. On the first day of the recording, the subjects were requested to fill a questionnaire, "Center of Epidemiologic Scale for Depression Scale (CES-D)," to evaluate the severity of their depressive state. RESULTS: There was a significant correlation between the DAL-M and DAL-A in both males (r=0.52, n=27) and females (r=0.50, n=21). TST was significantly correlated with depressive state only in the females (r=-0.49, n=28). In contrast, there was no significant correlation between the DAL and the severity of the depressive state. By multiple regression analysis, TST was only a good predictor of depressive state. CONCLUSION: The results of this investigation suggest that TST could be used as an index of the depressive state. The TST values in this study decreased as the severity of the depressive state increased, which suggests an association between the severity of the depressive state and insufficient sleep.

Activity Cycles↗
PubMed2006

Chlorpyrifos induces apoptosis in human monocyte cell line U937.

In order to investigate chlorpyrifos-induced cell death and its underlying mechanism in human immune cells, a human monocyte like cell line (U937) was treated with chlorpyrifos at 4.45-570microM for 0.5-24h at 37 degrees Celsius in a 5% CO(2) incubator. We first found that chlorpyrifos induced cell death of U937 in a dose- and time-dependent manner, as shown by LDH and MTT assays and PI uptake. Then, we investigated if chlorpyrifos-induced cell death consisted of apoptosis, as determined by analysis of Annexin-V staining and the intracellular level of active caspase-3 by flow cytometry, and DNA fragmentation analysis. We found that chlorpyrifos induced apoptosis in U937 in a time- and dose-dependent manner, as shown by Annexin-V staining. DNA fragmentation was detected when cells were treated with 71 to 284microM chlorpyrifos for 4 or 6h. Chlorpyrifos also induced an increase of intracellular active caspase-3 in U937 cells in a dose-dependent manner, and a caspase-3 inhibitor, Z-DEVD-FMK, significantly inhibited the chlorpyrifos-induced apoptosis. These findings indicate that chlorpyrifos can induce apoptosis in U937 cells, and this effect is partially mediated by activation of intracellular caspase-3.

Apoptosis↗
PubMed2006

EM703 improves bleomycin-induced pulmonary fibrosis in mice by the inhibition of TGF-beta signaling in lung fibroblasts.

BACKGROUND: Fourteen-membered ring macrolides have been effective in reducing chronic airway inflammation and also preventing lung injury and fibrosis in bleomycin-challenged mice via anti-inflammatory effects. EM703 is a new derivative of erythromycin (EM) without the bactericidal effects. We investigated the anti-inflammatory and antifibrotic effects of EM703 in an experimental model of bleomycin-induced lung injury and subsequent fibrosis in mice. METHODS: Seven-week-old male ICR mice were used. All experiments used eight mice/group, unless otherwise noted in the figure legends. Bleomycin was administered intravenously to the mice on day 0. EM703 was orally administered daily to mice. All groups were examined for cell populations in the bronchoalveolar lavage (BAL) fluid and for induction of messenger RNA (mRNA) of Smad3 and Smad4 in the lung tissues by reverse transcriptase (RT)-polymerase chainreaction (PCR) on day 7. Fibroblastic foci were assessed histologically, and the hydroxyproline content was chemically determined in the lung tissues on day 28. We performed assay of proliferation and soluble collagen production, and examined the induction of mRNA of Smad3 and Smad4 by RT-PCR in murine lung fibroblast cell line MLg2908. We also examined Smad3, Smad4 and phosphorylated Smad2/3 (p-Smad2/3) protein assay by western blotting in MLg2908. RESULTS: Bleomycin-induced lung fibrosis, and the infiltration of macrophages and neutrophils into the airspace were inhibited by EM703. The expression of Smad3 and Smad4 mRNA was clearly attenuated by bleomycin, but was recovered by EM703. EM703 also inhibited fibroblast proliferation and the collagen production in lung fibroblasts induced by Transforming growth factor-beta (TGF-beta). The expression of Smad3 and Smad4 mRNA in murine lung fibroblasts disappeared due to TGF-beta, but was recovered by EM703. EM703 inhibited the expression of p-Smad2/3 and Smad4 protein in murine lung fibroblasts induced by TGF-beta. CONCLUSION: These findings suggest that EM703 improves bleomycin-induced pulmonary fibrosis in mice by actions of anti-inflammation and regulation of TGF-beta signaling in lung fibroblasts.

Animals↗
PubMed2006

Phytoncides (wood essential oils) induce human natural killer cell activity.

To explore the effect of forest bathing on the human immune system, we investigated the effect of phytoncides (wood essential oils) on natural killer (NK) activity and the expression of perforin, granzyme A and granulysin in human NK cells. We used NK-92MI cell, an interleukin-2 independent human NK cell line derived from the NK-92 cell, in the present study. NK-92MI cells express the CD56 surface marker, perforin, granzyme A, and granulysin by flow cytometry and are highly cytotoxic to K562 cells in chromium release assay. Phytoncides significantly increase cytolytic activity of NK-92MI cells in a dose-dependent manner and significantly increase the expression of perforin, granzyme A, and granulysin in the NK-92MI cells. Phytoncides also partially, but significantly, restore the decreased human NK activity and the decreased perforin, granzyme A, and granulysin expression in NK-92MI cells induced by dimethyl 2,2-dichlorovinyl phosphate (DDVP), an organophosphorus pesticide. Pretreatment with phytoncides partially prevents DDVP-induced inhibition of NK activity. Taken together, these data indicate that phytoncides significantly enhance human NK activity and this effect is at least partially mediated by induction of intracellular perforin, granzyme A, and granulysin.

Antigens, Differentiation↗
PubMed2006

A monoclonal antibody to hippuric acid: an improved enzyme-linked immunosorbent assay for biological monitoring of toluene exposure.

A novel monoclonal antibody (MAb) to hippuric acid (HA) was prepared using an HA analog, N-alpha-benzoyl-lysine, as an immunogen. An enzyme-linked immunosorbent assay (ELISA) for HA was established using the anti-HA MAb named HA01BL. When the specificity of the MAb was analyzed by the ELISA system, the MAb was revealed to be less reactive to methylhippuric acids and to be more specific to HA than previously reported polyclonal antibodies. The detection limit of HA by the ELISA was approximately 1 microg/mL. The urinary HA concentration determined by the ELISA system correlated well with that obtained by high performance liquid chromatography.

Animals↗
PubMed2006

Factors associated with perceived health of very old inhabitants of Japan.

BACKGROUND: Perceived health or self-rated subjective health of the oldest inhabitants of Japan was studied to identify its associated impact on their lives. OBJECTIVE: The health status of the aged was evaluated to elucidate the correlation between their health complaints and perceived health status as expressed on a comprehensive rating scale. METHODS: A health questionnaire including the Todai Health Index (THI) was applied to 529 inhabitants of Central Japan, aged 85 years or older. THI is a symptom checklist composed of 130 questions. Twelve scale scores and two discriminant function values were calculated. Response rate was 99.8% (528/529). Other items included sex, age, prevalence of chronic disease and its treatment, marital status, academic career, and friendship. A question 'How is your health?' with five ordinal response options was used to assess perceived health. RESULTS: The mean values of THI scale scores of lie and aggression of poor (poor or extremely poor) perceived health were significantly lower than those of good (extremely good or good) perceived health. The mean values of other THI scale scores of poor perceived health except impulsiveness and nervousness in male subjects were significantly higher than those of good perceived health. Spearman's rank correlation coefficients between perceived health score and THI scale scores were all statistically significant. Furthermore, multiple logistic regression analysis was conducted to identify significant factors predicting perceived health. THI scale scores of physical symptoms, depressive state, and irregular daily life were significant. Odds ratios and 95% confidence intervals in parentheses on each factor were 1.15 (1.07-1.23), 1.14 (1.02-1.30), and 1.15 (1.02-1.30), respectively. CONCLUSIONS: Poor perceived health was significantly associated with irregular lifestyle, physical and mental complaints. Perceived health reflects the health status of the oldest inhabitants of Japan.

Aged↗
PubMed2006

Utility of second derivative of the finger photoplethysmogram for the estimation of the risk of coronary heart disease in the general population.

BACKGROUND: Increased arterial stiffness has been shown to be associated with coronary heart disease (CHD). However, it remains unclear as to whether the second derivative of the finger photoplethysmogram (SDPTG), a non-invasive method for the assessment of arterial stiffness, is useful for the estimation of risk of CHD in the general population. METHODS AND RESULTS: The SDPTG in 211 subjects (age: 63+/-15 years, range: 21-91 years, 93 males) was recorded without apparent atherosclerotic disorders from a community. The relationship between the SDPTG indices (b/a and d/a) and coronary risk factors (n=211) or the Framingham risk score (n=158, age: 60+/-12 years, range: 30-74 years, 63 males) were analyzed. The SDPTG indices significantly correlated with the Framingham risk score in both genders (b/a; r(male) =0.43, r(female) =0.54 and d/a; r(male) =-0.38, r(female) =-0.58), as well as several coronary risk factors. In the receiver operating characteristics curve analyses, the b/a discriminated high-risk subjects for CHD, who were in the highest quintile of the Framingham risk score in each gender, with a sensitivity and specificity of 0.85 and 0.58 in males and 0.83 and 0.72 in females, respectively. CONCLUSIONS: These results suggest that the SDPTG is useful for the estimation of risk of CHD in the general population.

Adult↗
PubMed2006

Relations of body mass index and coronary risk as estimated by the Framingham Risk Score.

The Framingham Risk Score is considered to predict 10-yr. risk of developing coronary heart disease. Other risk factors, such as a family history of coronary heart disease, sedentary lifestyle, and obesity should also be considered when estimating the risk of development of coronary heart disease. The last two factors can be modified by therapeutic lifestyle alterations. This cross-sectional coronary risk assessment of 16,871 Japanese subjects was estimated by the Framingham Risk Score. Sex and age were included in the calculation of the Framingham Risk Score. When multiple regression analysis was conducted controlling for age, regular physical exercise, smoking, and drinking, the body mass index was significantly associated with the Framingham Risk Score. The standardized regression coefficients for body mass index were .271 in men (p < .001) and .211 in women (p < .001), respectively. The significant association of body mass index with coronary heart disease risk, as estimated by the Framingham Risk Score, confirmed prior work.

Body Mass Index↗
PubMed2006

The mechanism of organophosphorus pesticide-induced inhibition of cytolytic activity of killer cells.

The main toxicity of organophosphorus pesticides (OPs) is neurotoxicity, which is caused by the inhibition of acetylcholinesterase. OPs also affect immune responses including effects on antibody production, IL-2 production, T cell proliferation, decrease of CD5 cells, and increase of CD26 cells and autoantibodies. However, there have been few papers investigating the mechanism of OP-induced inhibition of cytolytic activity of killer cells. This study reviews the new mechanism of OP-induced inhibition of activities of natural killer (NK), lymphokine-activated killer (LAK) and cytotoxic T lymphocytes (CTL). NK, LAK and CTL induce cell death in tumor or virus-infected target cells by two main mechanisms. The first mechanism is direct release of cytolytic granules that contain perforin, granzymes, and granulysin by exocytosis to kill target cells, which is called the granule exocytosis pathway. The second mechanism is mediated by the Fas ligand (Fas-L)/Fas pathway. To date, it has been reported that OPs inhibit NK, LAK and CTL activities by at least the following three mechanisms: 1) OPs impair the granule exocytosis pathway of NK, LAK and CTL cells by inhibiting the activity of granzymes, and by decreasing the intracellular level of perforin, granzyme A and granulysin, which was mediated by inducing degranulation of NK cells and by inhibiting the transcript of mRNA of perforin, granzyme A and granulysin; 2) OPs impair the FasL/Fas pathway of NK, LAK and CTL cells, as investigated by using perforin-knockout mice, in which the granule exocytosis pathway of NK cells does not function and only the FasL/Fas pathway remains functional; 3) OPs induce apoptosis of immune cells.

Animals↗
PubMed2005

Expression of enzymatically active human granzyme 3 in Escherichia coli for analysis of its substrate specificity.

Human granzyme 3 (Gr3) is a serine protease contained in the granules of natural killer cells and cytotoxic T lymphocytes. To elucidate the biochemical and physiological characteristics of Gr3, we attempted to prepare an enzymatically active recombinant human Gr3 without refolding and proteolytic activation. An expression vector was constructed, in which the pre-/pro-peptide coding sequence of Gr3 was replaced with the bacterial pelB leader sequence. The resultant expression product was a fully active protease in the periplasmic fraction of Escherichia coli and was purified to homogeneity. The purified enzyme effectively hydrolyzed Z-Lys-SBzl, a conventionally used substrate of Gr3. In addition, it also hydrolyzed the peptide substrate library FRETS-25Xaa series, required basic amino acid residues, Arg or Lys, at the P1 position, and most efficiently hydrolyzed the carboxylic side of Phe-Tyr-Arg downward arrow (P3-P2-P1) sequence of the 475 tripeptide combinations.

Amino Acid Sequence↗
PubMed2005

Dimethyl 2,2-dichlorovinyl phosphate (DDVP) markedly decreases the expression of perforin, granzyme A and granulysin in human NK-92CI cell line.

Natural killer (NK), lymphokine-activated killer (LAK) and cytotoxic T lymphocyte (CTL) cells kill target cells by the directed release of cytolytic granules that contain perforin, granzymes and granulysin. We previously have found that dimethyl 2,2-dichlorovinyl phosphate (DDVP), an organophosphorus pesticide significantly inhibited NK, LAK and CTL activities via the inhibition of granzyme activity. To further explore the mechanism of organophosphorus pesticide-induced inhibition of cell-mediated cytolysis, we asked here whether organophosphorus pesticides affect the expression of perforin, granzyme and granulysin in NK cells. We used NK-92CI cell, an interleukin-2 (IL-2) independent human NK cell line. We confirmed that NK-92CI cells express CD56 surface marker, perforin, granzyme A and granulysin by flow cytometry and immunofluorescence microscope, and that it is highly cytotoxic to K562 cells in chromium release assay. We found that DDVP significantly decreases the expression of perforin, granzyme A and granulysin in NK-92CI cells in a dose-dependent manner. Immunocytochemical results showed that DDVP significantly decreases perforin, granzyme A and granulysin positive granules in NK-92CI cell, which may be due to the degranulation. We also found that DDVP have a modest, but a significant inhibitory effect on the transcription of mRNA of perforin, granzyme A and granulysin.

Antigens, Differentiation, T-Lymphocyte↗
PubMed2005

The activation mechanism of human porphobilinogen synthase by 2-mercaptoethanol: intrasubunit transfer of a reserve zinc ion and coordination with three cysteines in the active center.

Human porphobilinogen synthase [EC.4.2.1.24] is a homo-octamer enzyme. In the active center of each subunit, four cysteines are titrated with 5,5'-dithiobis(2-nitrobenzoic acid). Cys(122), Cys(124) and Cys(132) are placed near two catalytic sites, Lys(199) and Lys(252), and coordinate a zinc ion, referred to as "a proximal zinc ion", and Cys(223) is placed at the orifice of the catalytic cavity and coordinates a zinc ion, referred to as "a distal zinc ion", with His(131) . When the wild-type enzymes C122A (Cys(122)-->Ala), C124A (Cys(124)-->Ala), C132A (Cys(132)-->Ala) and C223A (Cys(223)-->Ala) were oxidized by hydrogen peroxide, the levels of activity were decreased. Two cysteines were titrated with 5,5'-dithiobis(2-nitrobenzoic acid) in the wild-type enzyme, while on the other hand, one cysteine was titrated in the mutant enzymes. When wild-type and mutant enzymes were reduced by 2-mercaptoethanol, the levels of activity were increased: four and three cysteines were titrated, respectively, suggesting that a disulfide bond was formed among Cys(122), Cys(124) and Cys(132) under oxidizing conditions. We analyzed the enzyme-bound zinc ion of these enzymes using inductively coupled plasma mass spectrometry with gel-filtration chromatography. The results for C223A showed that the number of proximal zinc ions correlated to the level of enzymatic activity. Furthermore, zinc-ion-free 2-mercaptoethanol increased the activity of the wild-type enzyme without a change in the total number of zinc ions, but C223A was not activated. These findings suggest that a distal zinc ion moved to the proximal binding site when a disulfide bond among Cys(122), Cys(124) and Cys(132) was reduced by reductants. Thus, in the catalytic functioning of the enzyme, the distal zinc ion does not directly contribute but serves rather as a reserve as the next proximal one that catalyzes the enzyme reaction. A redox change of the three cysteines in the active center accommodates the catch and release of the reserve distal zinc ion placed at the orifice of the catalytic cavity.

Binding Sites↗
PubMed2005

Effect of electric foot shock and psychological stress on activities of murine splenic natural killer and lymphokine-activated killer cells, cytotoxic T lymphocytes, natural killer receptors and mRNA transcripts for granzymes and perforin.

To explore the mechanism of stress-induced inhibition of natural killer (NK) activity, female C57BL/6 mice were stimulated by electric foot shock and psychological stress for 7 days consecutively. The shocked mice received scrambled, uncontrollable, inescapable 0.6 mA electric shocks in a communication box 120 times during 60 min. The mice in the psychological stress group were put into the communication box without electric foot shock. The plasma corticosterone level in both stressed groups was significantly higher than that in controls on days 1, 3, 5 and 7 and showed the highest level on day 3 in the foot shock stress. According to these results, therefore, we investigated the effect of stress on immunological function on day 3, and measured body weight, weight of the spleen, number of splenocytes, splenic NK, lymphokine-activated killer (LAK) and cytotoxic T lymphocyte (CTL) activities, NK receptors, and mRNA transcripts for granzymes A and B and perforin in splenocytes. The NK, LAK and CTL activities, and NK receptors in mice with both types of stress were significantly decreased compared to those of the control mice, but the decreases were greater in the foot-shocked mice than in the psychological-stress mice. The mRNA transcripts for granzyme A and perforin were significantly decreased only in the foot-shocked mice. On the other hand, the foot-shock stress increased granzyme B. The above findings suggest that stress induced inhibition of NK, LAK and CTL activities partially via affecting NK receptors, granzymes and perforin.

Animals↗
PubMed2005

Hemolysis of erythrocytes by granulysin-derived peptides but not by granulysin.

Granulysin, a 9-kDa protein localized in human cytolytic T lymphocytes and natural killer cell granules, is cytolytic against tumors and microbes but not against red blood cells. Synthetic peptides corresponding to the central region of granulysin recapitulate the lytic activity of the intact molecule, and some peptides cause hemolysis of red blood cells. Peptides in which cysteine residues were replaced by serine maintain their activity against microbes but lose activity against human cells, suggesting their potential as antibiotics. Studies were undertaken to determine the mechanism of resistance of red blood cells to granulysin and sensitivity to a subset of granulysin-derived peptides. Granulysin lyses immature reticulocytes, which have mitochondria, but not red blood cells. Granulysin lyses U937 cells but not U937 cells lacking mitochondrial DNA and a functional respiratory chain (U937rho(o) degrees cells), further demonstrating the requirement of intact mitochondria for granulysin-mediated death. Peptide G8, which corresponds to helix 2/loop 2/helix 3, lyses red blood cells, while peptide G9, which is identical except that the cysteine residues were replaced by serine, does not lyse red blood cells. Granulysin peptide-induced hemolysis is markedly inhibited by an anion transporter inhibitor and by Na(+), K(+), and Ca(2+) channel blockers but not by Na(+)/K(+) pump, cotransport, or Cl(-) channel blockers. Although recombinant granulysin and G9 peptide do not induce hemolysis, they both competitively inhibit G8-induced hemolysis. The finding that some derivatives of granulysin are hemolytic may have important implications for the design of granulysin-based antimicrobial therapeutics.

Amino Acid Sequence↗
PubMed2005

[The trial of simple gas analysis of tobacco smoke that can be used for medical education].

OBJECTIVE: The purpose of this study was to check a simple sampling and easy gas analysis of tobacco smoke for effective tobacco intervention in medical education. METHODS: The mainstream of tobacco smoke was sampled by a syringe (50 ml) at five, ten and twenty seconds. The extracted mainstream was moved to a commercial PET bottle (2000 ml), and measured with gas detector tubes. The sidestream, which rises from the tip of the cigarette, was collected into a commercial PET bottle for a duration of 30 or 60 seconds. Formaldehyde, acetaldehyde, ammonia, hydrogen cyanide, and nitrogen oxides (NO, NO2) in the tobacco smoke were measured. Then, these gasses in the tobacco smoke of four brands of cigarettes were compared. This trial was conducted in third-year medical students, and the changes in attitudes to smokers and tobacco itself were investigated. RESULTS: The method of sampling 50 ml for 5 seconds produced the highest concentration of each gas in the mainstream. The gas concentration in the sidestream increased as the sampling time increased. The gas concentration in mainstream of "Lucia" was the highest of the used four brands, and the gas concentrations in the sidestream of "Mild Seven Prime" were higher than those of the other brands. Many medical students obtained knowledge about the toxicity of smoking by this experiment study. CONCLUSION: We studied a simple sampling method of tobacco smoke, and gas analysis with gas detector tubes. This method is recommended for tobacco education and intervention in medical education.

Acetaldehyde↗
PubMed2005

Insomnia as a sequela of sarin toxicity several years after exposure in Tokyo subway trains.

More than 5,000 passengers on Tokyo subway trains were injured with toxic chemicals including the nerve gas "sarin" on March 20, 1995. The purpose of this study was to identify the effect of sarin exposure on insomnia in a cross-sectional study. A self-administered questionnaire concerning sleep-related items was distributed to victims of sarin exposure in October and November, 2003. Questionnaires were completed by 161 of the 163 participants (98.8%), who were selected from 1,500 subjects. Among them, the authors selected 75 women 30 to 69 years of age. Control participants were collected from inhabitants living in Maebachi City, Gunma Prefecture, Japan. For the younger exposed group (under 50 yr. of age), percentages of poor sleep, difficulty falling asleep, intermittent awakening, early morning awakening, a feeling of light overnight sleep, and insomnia were significantly higher than those for the control group. In contrast, the older exposed group (ages 50 to 69 years) had significantly higher prevalence of poor sleep, a feeling of light overnight sleep, and early morning awakening for the exposed group when compared with the control group. The high prevalence of insomnia and insomnia-related factors for victims especially under 50 years of age suggests a need for research on sleep quality after sarin exposure. Although posttraumatic stress disorder is assumed to be a psychological effect of exposure to a toxic substance, a cause-and-effect relationship has not been established.

Adult↗