Role of autopsy imaging in veterinary forensic medicine: experiences in 39 cases.

While numerous scientific studies have suggested the usefulness of autopsy imaging (Ai) in the field of human forensic medicine, the use of imaging modalities for the purpose of veterinary forensics is currently scant. The current study performed Ai on suspicious dead animals requested by the police department to determine their cause of death. Radiography and/or computed tomography and/or magnetic resonance imaging were performed on 39 suspicious dead animals before necropsy. After diagnostic imaging, pathological examination was performed, with drug testing added as needed. Among the 39 cases, 28, 6, 3, 1, and 1 involved cats, dogs, rabbits, a ferret, and a pigeon, respectively. The major Ai findings included skull and rib fractures, subcutaneous emphysema, pneumothorax, pneumoperitoneum, diaphragmatic hernia, and abdominal rupture. The leading causes of death, determined comprehensively via Ai and pathological reports and drug test results, included traumatic impact, blood loss, poisoning, suffocation, tension pneumothorax, starvation, and drowning, all of which have been strongly suspected to indicate animal abuse by humans. All eight cases of skull fractures and all five cases of poisoning, including suspected poisoning, were of cats. As the numbers of dogs and cats in Japan are almost equal, violence against cats may occur more frequently than dogs. Ai can be a valuable examination tool for veterinary forensic cases. As computed tomography is useful for ruling out fractures that screening computed tomography before necropsy is a more practical option for veterinary forensics.

[In vitro screening of psychoactive drugs].

Prior to the designation of illegal drugs (psychoactive drugs) by prefectural regulations, the Tokyo Metropolitan Government conducts surveys on the risk of drugs, reports the results to the governor through the Tokyo Metropolitan Government Advisory Committee on Illegal Drugs, an affiliated organization, and provides the central government with information. The Tokyo Metropolitan Institute of Public Health conducts identification of the constituents of drugs and biological effect tests to help the committee analyze and assess information on the risks of drugs. Narcotics and stimulants increase the concentrations of dopamine, serotonin, and norepinephrine, i.e., neurotransmitters, in the presynaptic clefts, exerting an excitatory effect. In the postsynaptic region, these neurotransmitters are considered to directly combine with the receptors and activate guanine nucleotide-binding proteins, causing activation. We have developed nine types (categorized into three groups) of simple, high-throughput measurement systems and examined their measurement methods. The systems are designed to assess the following properties of drugs: effects of: 1) inhibiting reuptake; 2) stimulating the release of neurotransmitters in the presynaptic region; and 3) activating G proteins in the postsynaptic region. The systems provide useful information in that they allow searches for the effects of a variety of psychoactive substances that are expected to become widespread, e.g., designer drugs, hallucinogenic plants and synthetic cannabinoids; they also allow you to conduct a test using micrograms of a drug, facilitating testing even when it is not available in a large quantity.

[In vivo screening of illegal drug].

The neuro-behavioral observation scorebook that improved the previous observation methods of Irwin was followed, the test material was administered to 5 mice per each group, and the mean value of the obtained score was determined. The behavior of a normal animal was assumed to be point 0, animals showing suppressive behavior were scored in the minus region, and animals that showed excitement behavior were scored in the plus region. Each score was divided into three stages, according to the level of strength of the biological effect. The score of each observation item was totaled, and the level of the strength of the biological effect in the item was judged according to its mean value. These test methods of neuro-behavioral observations we proposed were able to detect the biological effects of a drug simply and promptly, and contributed sufficient data to support an administrative measure aimed at anticipating and improving the prevention of health damage in humans by non-regulated drugs from a scientific perspective. Recently, we developed a method of serial measurement of the quantity of monoamine in the mouse central nervous system by microdialysis, and performed it. The Kanagawa Prefectural Institute of Public Health conducted a study of the biological effect of non-regulated drugs. A characteristic here is what they examined about drug-dependency other than observing the behavior of the animal.

Serum level of expressed in renal carcinoma (ERC)/ mesothelin in rats with mesothelial proliferative lesions induced by multi-wall carbon nanotube (MWCNT).

Expressed in renal carcinoma (ERC)/mesothelin is a good biomarker for human mesothelioma and has been investigated for its mechanistic rationale during the mesothelioma development. Studies are thus ongoing in our laboratories to assess expression of ERC/mesothelin in sera and normal/proliferative/neoplastic mesothelial tissues of animals untreated or given potentially mesothelioma-inducible xenobiotics, by an enzyme-linked immunosorbent assay (ELISA) for N- and C-(terminal fragments of) ERC/mesothelin and immunohistochemistry for C-ERC/mesothelin. In the present paper, we intend to communicate our preliminary data, because this is the first report to show how and from what stage the ERC/mesothelin expression changes during the chemical induction of mesothelial proliferative/neoplatic lesions. Serum N-ERC/mesothelin levels were 51.4 +/- 5.6 ng/ml in control male Fischer 344 rats, increased to 83.6 +/- 11.2 ng/ml in rats given a single intrascrotal administration of 1 mg/kg body weight of multi-wall carbon nanotube (MWCNT) and bearing mesothelial hyperplasia 52 weeks thereafter, and further elevated to 180 +/- 77 ng/ml in rats similarly treated and becoming moribund 40 weeks thereafter, or killed as scheduled at the end of week 52, bearing mesothelioma. While C-ERC/mesothelin was expressed in normal and hyperplastic mesothelia, the protein was detected only in epithelioid mesothelioma cells at the most superficial layer. It is thus suggested that ERC/mesothelin can be used as a biomarker of mesothelial proliferative lesions also in animals, and that the increase of levels may start from the early stage and be enhanced by the progression of the mesothelioma development.

Increase in in utero exposure to a migrant, 4,4'-butylidenebis(6-t-butyl-m-cresol), from nitrile-butadiene rubber gloves on brain aromatase activity in male rats.

4,4'-Butylidenebis(6-t-butyl-m-cresol) (BBBC) can be eluted from disposable gloves made of nitrile-butadiene rubber and possibly also detected in food. It has been reported that BBBC is an androgen and estrogen antagonist in vitro. Previously, BBBC (1.0 mg/kg body weight (bw)/d) was subcutaneously administered to pregnant rats from gestation days 11 through 18, and the effects on male offspring (postnatal day 102) were examined. Altered levels and turnover of the monoamines dopamine, serotonin, and noradrenalin as well as their metabolites were detected. This report measured the level of serum testosterone following prenatal exposure to BBBC (0.1, 1.0, 10 mg/kg bw/d) in male rats, and measured aromatase activity of the hypothalamus-preoptic area with a close connection to the sexual differentiation and sexual behavior of BBBC-treated rat brains. The serum testosterone level rose depending on exposure, and aromatase activity of the basomedial nucleus of amygdale region was increased in the BBBC-treated group compared with the control. These results suggested that prenatal exposure to BBBC affects the central nervous system of male rat offspring, and BBBC may be an endocrine disrupting-chemical during the fetal period, and might influence the functional development of the brain.

The effects of in utero exposure to a migrant, 4,4'-butylidenebis(6-t-butyl-m-cresol), from nitrile-butadiene rubber gloves on monoamine neurotransmitter in rats.

4,4'-Butylidenebis(6-t-butyl-m-cresol) (BBBC) can be eluted from disposable gloves made of nitrile-butadiene rubber and possibly also detected in food. We have reported that BBBC is an androgen and estrogen antagonist. In this report, BBBC (0.1, 1.0 mg/kg body weight (bw)/day) was subcutaneously administered to pregnant rats from gestation days 11 through 18 and the effects on male offspring (postnatal day (PND) 102) were examined. Body weight at lactation and brain weight at PND 102 were decreased in the 1.0 mg/kg bw BBBC-treated group. Altered levels and turnover of the monoamines dopamine (DA), serotonin (5-HT), and noradrenalin (NA) as well as their metabolites were detected. In the prefrontal cortex DA, 3,4-dihydroxyphenylacetic acid (DOPAC), 5-HT, 5-hydroxyindole-3-acetic acid (5-HIAA) levels were significantly increased, but homovanillic acid (HVA)/DA was decreased. In the striatum NA level, DOPAC/DA and HVA/DA were significantly increased, but 3-methoxy-4-hydrophenylglycol hemipiperazinium (MHPG) level and MHPG/NA were decreased. In hippocampus MHPG level was significantly decreased. In hypothalamus 5-HIAA level and 5-HIAA/5-HT were significantly increased. These results suggested that prenatal exposure to BBBC affects the central nervous system of male rat offspring, and BBBC may be an endocrine disrupting-chemical during the fetal periods.

[In-vitro screening of psychoactive drugs to prevent abuses].

The Tokyo Metropolitan Government in Japan enacted an "Ordinance concerning the abuse prevention of the psychoactive drugs" in April 2006 that prohibited the manufacture, cultivation, sales, possession, use, etc., of these drugs. Therefore, we constructed a reproducible, simple, and small-scale determination method of the psychoactive drugs for the re-uptake and the release of monoamines (dopamine, serotonin and norepinephrine), and the activation of [(35)S]guanosine-5'-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins (G proteins). These assays were then applied to study the effects of different kinds (phenethylamine derivatives, tryptamine derivatives, and piperazine derivatives) of non-medically used psychoactive drugs on monoamine re-uptake and release, and G-protein binding. The results suggested that some drugs strongly act on the central nerve system to the same extent as the drugs. This assay system was able to designate psychoactive drugs as prohibited substances in accordance with criteria set forth by the Tokyo Metropolitan government.

In vitro screening assay for detecting aromatase activity using rat ovarian microsomes and estrone ELISA.

Aromatase, a key steroidogenic enzyme that catalyses the conversion of androgens to estrogens, present a target for endocrine disrupting chemicals. However, little is known about the effect of pollutants on aromatase enzymes. In this study, we first optimized a non-radioisotope aromatase assay using rat ovarian microsomes in vitro and measuring the estrone level with an enzyme-linked immunosorbent assay (EIA method). The sensitivity of the EIA method was about ten times as high as that of the radioisotope (RI) method. A significant positive correlation was indicated between EIA and RI method. We used this EIA assay system to investigate the effects of aromatase activity on 45 chemicals that had previously been reported to act as endocrine disruptors or to have the possibility of having such an effect. Six of the chemicals, rose bengal, erythrosine, phloxine, allura red, gallic acid, and tributyltin, inhibited aromatase activity. The inhibitory effect of rose bengal was the strongest (IC50=2.4 x 10(-8) M), and its strength of inhibition was about 100 times that of a known aromatase inhibitor, 4-hydroxy-androstenedione (IC50=2.6 x 10(-6) M) but was about 1/25 that of fadrazole (IC50=1.0 x 10(-9) M). It is thought that this EIA method would be useful for measuring the aromatase activity of microstructures.

Endocrine disruptive effects of chemicals eluted from nitrile-butadiene rubber gloves using reporter gene assay systems.

Disposable gloves made of nitrile-butadiene rubber (NBR) are used for contact with foodstuffs rather than polyvinyl chloride gloves containing di(2-ethylhexyl)phthalate (DEHP), because endocrine-disruptive effects are suspected for phthalate diesters including DEHP. However, 4,4'-butylidenebis(6-t-butyl-m-cresol) (BBBC), 2,4-di-t-butylphenol, and 2,2,4-trimetyl-1,3-pentanediol diisobutyrate can be eluted from NBR gloves, and possibly also detected in food. In this study, we examined the endocrine-disrupting effects of these chemicals via androgen receptor (AR) and estrogen receptor (ER)-mediated pathways using stably transfected reporter gene cell lines expressing AR (AR-EcoScreen system) and ER (MVLN cells), respectively. We also examined the binding activities of these chemicals to AR and ER. The IC50 value of BBBC for antagonistic androgen was in the range of 10(-6)M. The strength of inhibition was about 5 times that of a known androgen antagonist, 1,1'-(2,2-dichloroethylidene)bis[4-chlorobenzene] (p,p'-DDE), and similar to that of bisphenol A. The IC50 value of BBBC for antagonistic estrogen was in the range of 10(-6)M. These results suggest that BBBC and its structural homologue, 4,4'-thiobis(6-t-butyl-m-cresol) are androgen and estrogen antagonists. It is therefore necessary to study these chemicals in vivo, and clarify their effect on the endocrine system.

In vitro screening of psychoactive drugs by [(35)S]GTPgammaS binding in rat brain membranes.

We constructed a reproducible, simple, and small-scale determination method of the psychoactive drugs that acted directly on the monoamine receptor by measuring the activation of [(35)S]guanosine-5'-O-(3-thio)-triphosphate binding to guanine nucleotide-binding proteins (G proteins). This method can simultaneously measure the effects of three monoamines, namely dopamine (DA), serotonin (5-HT), and norepinephrine (NE), in rat brain membranes using a 96-well microplate. Activation of D(1) and D(2) receptors in striatal membranes by DA as well as 5-HT and NEalpha(2) receptors in cortical membranes could be measured. Of 12 tested phenethylamines, 2,5-dimethoxy-4-chlorophenethylamine (2C-C), 2,5-dimethoxy-4-ethylphenethylamine (2C-E), and 2,5-dimethoxy-4-iodophenethylamine (2C-I) stimulated G protein binding. The other phenethylamines did not affect G protein binding. All 7 tryptamines tested stimulated G protein binding with the following rank order of potency; 5-methoxy-N,N-dimethyltryptamine (5-MeO-DMT)>5-methoxy-N,N-diallyltryptamine (5-MeO-DALT)>5-methoxy-alpha-methyltryptamine (5-MeO-AMT)>or=5-methoxy-N,N-methylisopropyltryptamine (5-MeO-MIPT)>5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT)>N,N-dipropyltryptamine (DPT)>or=alpha-methyltryptamine (AMT). This assay system was able to designate psychoactive drugs as prohibited substances in accordance with criteria set forth by the Tokyo Metropolitan government.

Effects of oseltamivir phosphate (Tamiflu) and its metabolite (GS4071) on monoamine neurotransmission in the rat brain.

As abnormal behaviors such as jumping and falling from balcony were reported in patients aged 10 to 19 years who administrated oseltamivir phosphate (Tamiflu) for treatment influenza infection, the Ministry of Health, Labor and Welfare in Japan notified that, as a rule, Tamiflu should not be prescribed to patients aged 10 to 19 years. To examine the relationship between Tamiflu and abnormal behaviors, we investigated the effects of Tamiflu and its carboxylic acid metabolite, GS4071, on the central nervous system, that is, on 3 neurotransmitters (dopamine, serotonin, and norepinephrine) in presynapses (inhibition of re-uptake, promotion of release) and postsynapses (guanosine 5'-triphosphate (GTP) gammaS binding), using rat brain synaptosomes. Neither Tamiflu nor GS4071 influenced the re-uptake/release of the 3 monoamines or GTP binding in postsynapses.

Effect of sacral magnetic stimulation on the anorectal manometric activity: a new modality for examining sacro-rectoanal interaction.

We examined the interaction between the sacral neural system and the anorectal activity using a technique comprising repetitive magnetic stimulation. Thirteen control children without any bowel dysmotility (age range; 3 month old to 15 year old), 20 patients with chronic constipation (1 month old to 14 year old), and nine pre-operative patients with Hirschsprung's disease (1 month old to 6 year old) were examined. Magnetic stimulation was applied at the S3 level using the MagPro (Medtronic) device while simultaneously performing manometric recordings of the anorectal activity. In the 13 controls and the 20 patients with chronic constipation, the rectoanal reflex was demonstrated by balloon rectal dilatation. The sphincter response to magnetic stimulation was biphasic in the controls, consisting of an initial rise followed by a decrease in the sphincter pressure, while it varied among the patients with chronic constipation including a biphasic response in 16 patients, no response in three patients, and only a transient rise in pressure in one patient. In nine pre-operative patients with Hirschsprung's disease, no rectoanal reflex was observed, however, the sphincter pressure increased due to magnetic stimulation in six patients, while three patients exhibited no recordable responses. These results suggest that the repetitive magnetic stimulation technique is a valuable modality for investigating the neural interaction between the sacral nervous system and the anorectum.

Is mobile testis a true pathological condition due to a gubernaculum abnormality?

The present study was conducted to clarify whether a gubernaculum abnormality exists in mobile testis or not. Testicular fixation was performed in 57 patients with mobile testis with a mean age of 3.6 years (range; 7 months-12 years). Twenty-six patients were bilateral and 31 patients were unilateral cases. Control gubernaculum findings were obtained from 12 patients with hydrocele testis with a mean age of 3.3 years (1.6-5 years). In 12 patients with hydrocele testis, the gubernaculum was found from the lower third of the testis to the scrotal base inside the tunica vaginalis (plica gubernaculi), while outside the tunica vaginalis pars infravaginalis gubernaculi further extended into the scrotal base. On the other hand, in 57 patients with mobile testis, the gubernaculum was easily identified only inside the tunica vaginalis without any recognizable gubernaculum outside the tunica vaginalis. In 31 unilateral patients, associated anomalies of the contralateral testis were found in 13 patients (42%); 11 cases with undescended testis and two cases with ectopic testis. These results suggest that mobile testis might therefore be a true pathological condition, which were highly associated with contralateral testicular morbidities.

Effects of prenatal exposure to styrene trimers on genital organs and hormones in male rats.

Styrene trimers migrate from polystyrene food container into foods. We evaluated the estrogenic activity of styrene trimers such as 2,4,6-triphenyl-1-hexene (ST-1), 1a-phenyl-4a-(1'-phenylethyl)tetralin (ST-2), 1a-phenyl-4e-(1'-phenylethyl)tetralin(ST-3), 1e-phenyl-4a-(1'-phenylethyl)tetralin (ST-4), and 1e-phenyl-4e-(1'-phenylethyl)tetralin (ST-5) using the reporter-gene assay with MVLN cells stably expressing the estrogen-stimulated reporter gene, and it was confirmed that ST-1, ST-3, and ST-4 had estrogen-like activity. On the other hand, ST-2 and ST-5 had anti-estrogen-like activity. We examined the estrogenic activity in vivo of ST-1, ST-3, and ST-4. The styrene trimers were administered to pregnant rats, and the effects on the offspring were examined. ST-1, ST-3, or ST-4 (0, 10, 100, 1000 microg/kg body wt/day) were subcutaneously injected into pregnant rats from gestational Day 11 through 17, and the male offspring were sacrificed on postnatal days (PND) 101-103. In the ST-4 treatment groups, the relative anogenital distance on PND 3 was significantly shortened. The relative testis weight was remarkably decreased in all styrene trimer treatment groups. Relative weights of the prostate and epididymides significantly decreased in the ST-4 treatment groups. The relative brain weight was markedly reduced in the ST-3 and ST-4 treatment groups. A significant decrease of the Sertoli cell count was observed in the ST-1 and ST-4 treatment groups. The serum follicle stimulating hormone level was remarkably reduced in all styrene trimer treatment groups. The luteinizing hormone level was significantly decreased and the testosterone level increased in the ST-1 and ST-4 groups. These results suggest that prenatal exposure to estrogenic styrene trimers at low levels obstructed genital organ development, and disrupted the endocrine systems of male rat offspring.

Array comparative genomic hybridization analysis in first-trimester spontaneous abortions with 'normal' karyotypes.

Array comparative genomic hybridization (array CGH) analysis was conducted in chorionic villous samples from 20 first-trimester spontaneous abortions with G-banding normal chromosomes. A microarray, containing 2,173 BAC clones and covering the whole genome with a 1.5-Mb resolution, was constructed and used in the analysis. Two deletions were identified: a 1.4-Mb deletion at 3p26.2-p26.3 and a 13.7-Mb deletion at 13q32.3-qter. Reexamination of chromosome preparations from the sample with the 13.7-Mb deletion documented a mixture of cells with the 13q- chromosome and those with 46,XX chromosomes, the latter of which are likely to have been derived from contaminating decidual cells. This left the 1.4-Mb 3p deletion as the only instance with submicroscopic imbalance detected, giving a frequency of 1 in 19 (5%) G-banding normal abortions.

Effects of N-nitrosofenfluramine, a component of Chinese dietary supplement for weight loss, on CD-1 mice.

Many cases of hepatopathy including deaths have frequently occurred after ingestion of Chinese dietary supplements for weight loss containing N-nitrosofenfluramine (N-fen), a nitroso derivative of fenfluramine (Fen), which was used for the treatment of obesity in the United States. Since Fen decreases appetite by decreasing the serotonin level and exhibits an antibiotic effect, N-fen may have been added, expecting a similar effect. Thus, we synthesized N-fen and orally administered it to mice, and investigated its effect on the liver as well as on the cerebral serotonin nervous system to investigate whether N-fen exhibits an anorectic effect. Three doses of N-fen were orally administered once daily to mice for 1 week. No significant changes in body weight, food intake, and general condition were noted. The liver and kidney weights were significantly increased. On blood chemistry, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities were increased, and total bilirubin and albumin were slightly decreased. On histopathological examination, acidophilic changes and mild cellular swelling were noted in the liver. The liver drug-metabolizing enzyme (P-450) level was significantly higher. The effect of N-fen on the serotonin (5HT) nervous system was examined by quantitative autoradiography of the mouse brain, and it was found that N-fen did not decrease the 5HT nerve activity. Effects of reuptake and release of monoamine neurotransmitters [dopamine (DA), 5HT, and norepinephrine (NE)] were investigated. N-fen inhibited a little 5HT reuptake, and did not inhibit reuptakes of DA and NE. Moreover, N-fen did not affect release of the three monoamines. The above findings suggested that N-fen did not exhibit a serotonin nerve fiber-mediated anorectic effect in mice, but induced hepatopathy.

[Identification of Aloe species by random amplified polymorphic DNA (RAPD) analysis].

Juice and integument of leaves of 3 Aloe species, Aloe vera, A. ferox and A. africana, are not allowed to be used as food according to the Pharmaceutical Affairs Law in Japan. On the other hand, whole leaves of A. arborescens can be used as food. The present study was designed to distinguish Aloe species by random amplified polymorphic DNA (RAPD) analysis. DNA was isolated from fresh and dried leaves of the 4 Aloe species. Five out of 32 different 10-mer primers examined were useful for analysis. By comparison of the characteristic bands of PCR products on agarose gel, it was possible to distinguish the 4 species. Thus, the botanical species of Aloe in commercial food products can be identified by RAPD analysis.

Inhibition of Na(+),K(+)-ATPase by the extract of Stephania cephararantha HAYATA and bisbenzylisoquinoline alkaloid cycleanine, a major constituent.

The Stephania cephararantha HAYATA extract, and its constituent bisbenzylisoquinoline alkaloids, such as cycleanine, cepharanthine, isotetrandrine, berbamine, homoaromoline, and cepharanoline were studied for effects on Na(+),K(+)-ATPase activity. The S. cephararantha HAYATA extract inhibited Na(+),K(+)-ATPase activity with an apparent IC(50) value of 540 microg/mL. Cycleanine markedly inhibited Na(+),K(+)-ATPase activity with an IC(50) value of 6.2 x 10(-4)M. It slightly inhibited Mg(2+)-ATPase, H(+)-ATPase, and Ca(2+)-ATPase. No effects on alkaline and acid phosphatase activities were observed. The inhibition by isotetrandrine, homoaromoline, cepharanthine, and berbamine was less marked, and cepharanoline showed no effect. Five synthetic analogues of cepharanthine slightly inhibited the activity. The mechanism of inhibition by cycleanine on Na(+),K(+)-ATPase activity was examined in detail, and the following results were obtained in the overall reaction: (1) the mode of inhibition was noncompetitive with respect to ATP; (2) the degree of inhibition was decreased with a decrease of K(+) concentration; (3) it was not affected by Na(+) concentration; (4) the inhibition mechanism was different from that of ouabain. The activity of K(+)-dependent p-nitrophenyl phosphatase, a partial reaction of Na(+),K(+)-ATPase, did not appear to have been inhibited by cycleanine in the reaction mixture containing 15 mM K(+) (optimum condition). However, cycleanine increased the K(0.5) value for K(+) and reduced the K(i) values for Na(+) and ATP, in K(+)-dependent p-nitrophenyl phosphatase. Cycleanine might interact with the enzyme in Na.E(1)-P form and prevents the reaction step from Na.E(1)-P to E(2)-P.