Traumatic deltoid rupture caused by seatbelt during a traffic accident: a case report.

A right-handed 53-year-old man presented with a subcutaneous bruise on his right shoulder caused by a seatbelt during a traffic accident. He had no history of shoulder pain or hydrocortisone injections. The contour of the anterior deltoid was deformed and its belly was retracted distally. The active range of movement of the shoulder was limited to 120 degrees and the strength weakened to 3/5. Magnetic resonance imaging demonstrated detachment of the anterior fibres of the deltoid. Surgical repair of the deltoid and supraspinatus tendon was performed 2 months later using a pull-out suture technique. After 12 months of follow-up, the patient had returned to work without any problems. Both the range of movement and muscle strength had recovered completely.

PAC1 receptor localization in a model nervous system: light and electron microscopic immunocytochemistry on the earthworm ventral nerve cord ganglia.

The presence and pattern of pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptors were identified by means of pre- and post-embedding immunocytochemical methods in the ventral nerve cord ganglia (VNC) of the earthworm Eisenia fetida. Light and electron microscopic observations revealed the exact anatomical positions of labeled structures suggesting that PACAP mediates the activity of some interneurons, a few small motoneurons and certain sensory fibers that are located in ventrolateral, ventromedial and intermediomedial sensory longitudinal axon bundles of the VNC ganglia. No labeling was located on large interneuronal systems such as dorsal medial and lateral giant axon systems and ventral giant axons. At the ultrastructural level labeling was mainly restricted to endo- and plasma membranes showing characteristic unequal distribution in various neuron parts. An increasing abundance of PAC1 receptors located on both rough endoplasmic reticulum and plasma membranes was seen from perikarya to neural processes, indicating that intracellular membrane traffic might play a crucial role in the transportation of PAC1 receptors. High number of PAC1 receptors was found in both pre- and postsynaptic membranes in addition to extrasynaptic sites suggesting that PACAP acts as neurotransmitter and neuromodulator in the earthworm nervous system.

New scaffold for recombinant human bone morphogenetic protein-2.

A bioactive and resorbable scaffold is necessary to exhibit the osteoinductive potency of recombinant human bone morphogenetic protein-2 (rhBMP-2). In a previous study, we found that synthetic octacalcium phosphate (OCP) enhances bone regeneration and is replaced by newly formed bone after it is resorbed. We hypothesized that OCP may be useful as an effective scaffold for rhBMP-2 to enhance bone regeneration. To test this hypothesis, the present study was designed to investigate whether an OCP/BMP composite implant could more effectively enhance bone regeneration. A critical-sized defect was made in a rat calvarium and 1. 15 mg of OCP combined with 10 microg of rhBMP-2 (OCP/BMP 10 microg), 2. 15 mg of OCP combined with 1 microg of rhBMP-2 (OCP/BMP 1 microg), or 3. OCP (OCP alone) was implanted into the defect and fixed at 4 or 8 weeks after implantation. The percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. A statistical analysis showed that n-Bone% with OCP/BMP was significantly higher than that with OCP at both time points, whereas the difference in n-Bone% between OCP/BMP 10 microg and OCP/BMP 1 microg was not significant. The present results suggest that OCP can be used as an effective scaffold for rhBMP-2 and this OCP delivery system may be able to reduce the standard effective dose of rhBMP-2, which would be beneficial because low doses (<100 microg/g OCP) of rhBMP-2 enhance bone regeneration.

The activity ratio of the cytosolic MDH/LDH and the isoenzyme pattern of LDH in the peripheral leukocytes of dogs, cats and rabbits.

The activities of malate dehydrogenase (MDH) and lactate dehydrogenase (LDH) and the pattern of the isoenzymes of LDH were determined in the peripheral blood leukocytes of dogs, rabbits and cats. Rabbits had significantly higher plasma glucose concentrations than dogs or cats. Feline leukocytes showed higher LDH and lower MDH activities than canine or rabbit leukocytes. The M/L ratio, defined as the MDH activity divided by the LDH activity in cytosolic fractions, was considered to be a good indicator with which to evaluate the metabolic state in animal tissues. The M/L ratio was highest in canine and lowest in feline leukocytes. LDH-2 and LDH-3 isoenzymes were dominant in canine leukocytes. LDH-1 and LDH-2 were dominant in rabbit leukocytes, whereas LDH-5 was dominant in feline leukocytes. It was evident that there were significant differences in energy metabolism between the leukocytes of dogs, rabbits and cats.

CRBP suppresses breast cancer cell survival and anchorage-independent growth.

We showed earlier that cellular retinol-binding protein (CRBP) expression is downregulated in a subset of human breast cancers. We have now investigated the outcome of ectopic CRBP expression in MTSV1-7 cells, a SV40 T antigen-transformed human breast epithelial cell line devoid of endogenous CRBP expression. We found that: (i) CRBP did not inhibit adherent cell growth but suppressed foci formation in post-confluent cultures and colony formation in soft agar; (ii) this effect was due to CRBP inhibition of cell survival, as demonstrated by viability and TUNEL assays of cells in soft-agar or plated on polyHEMA-coated dishes; (iii) CRBP inhibited protein kinase B/Akt activation in cells in suspension but not in adherent cells and the CRBP suppression of anchorage-independent growth was mimicked by cell treatment with the phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002; (iv) CRBP enhanced retinyl ester formation and storage but did not regulate retinoic acid synthesis or retinoic acid receptor activity. Ectopic CRBP-mediated inhibition of anchorage-independent cell survival and colony formation in the absence of significantly altered responses to either retinol or retinoic acid was also documented in T47D human breast cancer cells. In conclusion, the data suggest two novel and linked CRBP functions in mammary epithelial cells: inhibition of the PI3K/Akt survival pathway and suppression of anchorage-independent growth.

Expression of beta-synuclein in normal human astrocytes.

Although alpha- and beta-synucleins are expressed predominantly in presynaptic nerve terminals, recent studies have demonstrated that alpha-synuclein is also expressed in cultured astrocytes and oligodendrocytes. We determined whether beta-synuclein might be expressed in astrocytes. Beta-synuclein mRNA and protein were detected in normal human astrocytes in culture, and immunofluorescent staining showed that beta-synuclein protein was expressed within the cytoplasm and nucleus. Furthermore, beta-synuclein immunoreactivity was present in astrocytes, but not in oligodendrocytes, in normal human brain tissues. Ultrastructurally, beta-synuclein immunoreactivity was found in the cytoplasm of astrocytes, in association with the plasma membrane, ribosomes, rough endoplasmic reticulum and the nuclear outer membrane. The novel expression of beta-synuclein in astrocytes may provide an important insight about the role of this protein.

Implantation of octacalcium phosphate combined with transforming growth factor-beta1 enhances bone repair as well as resorption of the implant in rat skull defects.

In our previous study, we reported that synthetic octacalcium phosphate (OCP) enhances bone repair if implanted in rat skull defects. We hypothesized that OCP can be used as an effective carrier for transforming growth factor-beta1 (TGF-beta1) to promote bone repair. We designed the present study to investigate histomorphometrically whether combination with recombinant human TGF-beta1 could promote bone repair caused by OCP per se (Control/OCP). A full-thickness standardized trephine defect was made in the rat parietal bone and OCP combined with recombinant human TGF-beta1 (TGF-beta1/OCP) or Control/OCP was implanted into the defect. Four rats from each group were fixed at 2, 4, and 8 weeks after implantation. Histomorphometrical analysis of the percentage of newly formed bone (n-Bone %) and remaining implants (r-Imp %) in the defect was performed. The statistical analysis showed the n-Bone % of TGF-beta1/OCP was significantly higher than that of the Control/OCP in week 4, whereas the r-Imp % of TGF-beta1/OCP was significantly lower than that of the Control/OCP. The present study demonstrated that OCP can be used as an effective carrier for TGF-beta1 and their combination enhances bone repair as well as resorption of the carrier OCP in the early stage of bone formation.

Osteoblastic differentiation of periosteum-derived cells is promoted by the physical contact with the bone matrix in vivo.

The periosteum contains osteoprogenitors that differentiate to osteoblasts in bone growth or repair. Our previous studies suggested the hypothesis that the physical contact of the periosteum with the bone matrix is requisite for the differentiation of osteoblasts. To test the hypothesis, the present study was designed to investigate how the contact between the periosteum and the bone matrix influences the osteoblastic differentiation of periosteal cells with establishing a new experimental model in vivo. Differentiation of osteoblasts was assessed by gene expression of type I collagen, osteocalcin and bone sialoprotein using in situ hybridization. A barrier was designed to prevent periosteal cells from contacting the bone matrix using the membrane filter. The membrane filter was inserted surgically between the surface of rat parietal bone and the periosteum after being punched out with pin holes. Periosteal cells were allowed to contact with the bone surface only through the pin holes. The pin hole was filled with cells derived from the periosteum 1 week after inserting the filter. Differentiation of osteoblasts in week 2 and noticeable bone formation in week 3 were identified on the bone surface only under the pin hole but not under the filter. The present study demonstrated that the physical contact with the bone matrix promotes osteoblastic differentiation of periosteum-derived cells in vivo.

Induction of drug metabolism-related enzymes by methylcholanthrene and phenobarbital in transgenic mice carrying human prototype c-Ha-ras gene and their wild type littermates.

Transgenic mice hemizygously carrying human c-Ha-ras proto-oncogene, Tg-rasH2 show very sensitive and facilitated carcinogenicity to various carcinogens. In this study, activities of certain enzymes related to drug metabolism and energy metabolism were measured in microsome and cytosol fractions of livers of Tg-rasH2 mice and their wild type littermates with both sexes treated with 3-methylcholanthrene (MC) and phenobarbital (PB). Aminopyrine N-demethylase activities increased significantly in livers of all mice treated with PB. MC and PB treatments induced significant increases in activities of UDP-glucuronosyltransferase and S-adenosyl homocysteinase compared to those in the non-treated groups in microsome fractions from all mice. In cytosol fractions of livers of all mice, glutathione S-transferase activity was significantly induced in the PB treated groups. There were no significant differences in activities of lactate dehydrogenase, glucose 6-phosphate dehydrogenase, pyruvate kinase and glucose 6-phosphatase related to energy metabolism in livers and kidneys among all mice. Tg-rasH2 mice showed stable activities of enzymes related to drug detoxication and energy metabolism similar to those of non-transgenic mice. These results suggest that the human c-Ha-ras transgene may not affect drug metabolism-related enzymes, and the facilitated carcinogenic response in the Tg-rasH2 mouse is not due to these enzymatic disorders.

Cosecretion of prolactin and growth hormone by dispersed pituitary cells of the adult bullfrog, Rana catesbeiana.

The coexistence of prolactin (PRL) and growth hormone (GH) was previously demonstrated in newly hatched bullfrog (Rana catesbeiana) tadpoles, whereas in adult bullfrogs, there were no cells containing both PRL and GH. However, a cell blot assay with enzymatically dispersed adult pituitary cells demonstrated the existence of cells secreting both PRL and GH. The number of cells secreting both PRL and GH was reduced by a protein synthesis inhibitor, cycloheximide, but not by an RNA synthesis inhibitor, actinomycin D. In situ hybridization and immunostaining of intact pituitary glands revealed the existence of GH mRNA in some of the PRL-immunoreactive cells and of PRL mRNA in some of the GH-immunoreactive cells. We propose that dispersion of the pituitary cells triggered the translation of GH mRNA in the PRL cells and/or of PRL mRNA in the GH cells.

Apoptosis induced by arsenic trioxide in leukemia U937 cells is dependent on activation of p38, inactivation of ERK and the Ca2+-dependent production of superoxide.

The mechanism of the induction of apoptosis by arsenic trioxide (As2O3), which was demonstrated recently to be an effective inducer of apoptosis in patients with leukemia, was examined in detail in human leukemia U937 cells. Upon treatment of U937 cells with 50 microM of As2O3, complete inactivation of the kinases ERK1 and ERK2 was detected within 30 min. p38 was activated within 3 hr, and the maximum activity was detected at 6 hr, when DNA fragmentation remained undetectable. Experiments with transfected cells that expressed constitutively activated MEK1 and a specific inhibitor of p38 also suggested that inactivation of ERKs and activation of p38 might be associated with the induction of apoptosis by As2O3. In contrast to the inactivation of ERKs and the activation of p38, activation of JNK by As2O3 appeared to protect cells against the induction of apoptosis. Treatment of U937 cells with As2O3 also caused the Ca2+-dependent production of superoxide and intracellular acidification and a decrease in the mitochondrial membrane potential at the early stages of induction of apoptosis by As2O3. These changes preceded the release of cytochrome c from mitochondria and the activation of caspase-3. It should be possible to exploit the unusual characteristics of the mechanism of induction of apoptosis by As2O3 in U937 cells by making use of synergistic effects of this compound with other inducers of apoptosis.

Atp-binding cassette transporter ABC2/ABCA2 in the rat brain: a novel mammalian lysosome-associated membrane protein and a specific marker for oligodendrocytes but not for myelin sheaths.

We recently cloned a full-length cDNA of the rat ATP-binding cassette transporter 2 (ABC2, or ABCA2) protein, a member of the ABC1 (or ABCA) subfamily (-ABC1/ABCA1 is a causal gene for Tangier disease) and found it to be strongly expressed in the rat brain. In this study, we identified ABC2 as a lysosome-associated membrane protein that is being localized specifically in oligodendrocytes. The ABC2-immunolabeled cells were detected mainly in the white matter but were also scattered in gray matter throughout the whole brain. In addition, these cells were found to be colocalized with 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) immunoreactivity when the marker antibody for oligodendrocytes was used. However, no such colocalization was observed with markers for other kinds of glial cells. Unlike the CNP antibody, which also intensely stains myelin sheaths in the white matter, ABC2 immunoreactivity was detected only in the cell bodies of oligodendrocytes. At the ultrastructural level, ABC2 immunoreactivity was detected mostly around lysosome and partly in Golgi apparatus by electron microscopy. This was confirmed by immunocolocalization of ABC2 and lysosomal markers in a neuroblastoma cell line. Immunoblotting analysis of ABC2 from the whole brain and the ABC2-transfected cell line revealed bands at approximately 260 kDa. The result of in situ hybridization with a riboprobe for ABC2 matched the results obtained from immunostaining. These findings strongly suggest that ABC2 is a specific marker for oligodendrocytes but not for myelinsheaths and that it is as a novel mammalian lysosome-associated membrane protein involved in myelinization or other kinds of metabolism in the CNS.

Effect of prolactin and androgen on the expression of the female-attracting pheromone silefrin in the abdominal gland of the newt, Cynops ensicauda.

Silefrin is a sodefrin-like, female-attracting pheromone comprising 10 amino acids that was isolated from the abdominal gland of the sword-tailed newt, Cynops ensicauda. Hormonal effects on the silefrin precursor mRNA expression and silefrin content in the abdominal gland were investigated in the present study by using Northern blot analysis and radioimmunoassay, respectively. In the abdominal gland of newts treated with prolactin (PRL) plus testosterone propionate (TP), silefrin precursor mRNA expression was markedly enhanced as compared with that in the newts injected with saline, PRL, or TP. Values for radioimmunoassayable silefrin content in the abdominal gland paralleled those for the silefrin precursor mRNA levels. Moreover, silefrin precursor mRNA signals, as revealed by in situ hybridization, as well as stainability of immunoreactive silefrin were much more intense in the epithelial cells of the abdominal gland of the PRL-plus-TP-treated animals than in those of controls. We thus conclude that PRL and androgen are important factors for enhancing silefrin synthesis.

The mechanism of geranylgeraniol-induced apoptosis involves activation, by a caspase-3-like protease, of a c-jun N-terminal kinase signaling cascade and differs from mechanisms of apoptosis induced by conventional chemotherapeutic drugs.

In the present study, we investigated the effects of geranylgeraniol (GGO), a potent inducer of apoptosis in various lines of human tumor cells, on signal transduction cascades involved in apoptosis in human leukemia cells. GGO strongly induced the activation of c-Jun N-terminal kinase (JNK/SAPK) within 2 h in U937 and K562 cells, while neither ERK nor p38 was activated to any considerable extent during GGO-induced apoptosis. Transient expression of a constitutively active mutant form of mitogen-activated protein kinase kinase 1 (MEKK1), deltaMEKK1, or of deltaMEKK1-green fluorescent protein (GFP) in K562 cells activated JNK, but not a caspase-3-like protease, and was insufficient to induce cell death but rendered cells susceptible to GGO-induced cell death. Stable expressions of deltaMEKK1-GFP in U937 cells gave similar results. In contrast to VP-16-induced apoptosis, GGO-induced activation of JNK was almost completely inhibited by benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD) and by benzyloxycarbonyl-Asp-CH2OC[O]-2,6,-dichlorobenzene (Z-Asp), indicating that the JNK-activation step is located downstream of the caspase signaling pathway in GGO-induced apoptosis. Moreover, apoptosis induced by GGO was significantly inhibited in two lines of cells with a dominant-negative deletion mutation in c-Jun, indicating a requirement for JNK signaling. In addition, unlike the effects on other inducers of apoptosis, the activation of JNK and of the caspase-3-like protease by GGO was significantly delayed by 12-O-tetradecanoylphorbol-13-acetate (TPA), suggesting that the site of inhibition by TPA might be located upstream of the protease and JNK in the GGO-induced apoptotic signaling pathway.

Splice variants of PAC(1) receptor during early neural development of rats.

The specific pituitary adenylate cyclase-activating polypeptide (PACAP) receptor, PAC(1)-R, consists of at least seven isoforms, and they are differentially coupled to signal transduction pathways by alternative splicing. We have found that the major splice variants of the PAC(1) receptor seen during development are the short splice isoform, PAC(1)-R-s (which does not contain either the "hip" or "hop" cassette), and another form, PAC(1)-R-hop (which contains the "hop" cassette). We also have applied an innovative molecular histochemical technique, in situ reverse transcription-polymerase chain reaction (RT-PCR), and determined that these two splice isoforms are colocalized in the neuroepithelia from the primitive streak stage.

Serum total bile acid level as a sensitive indicator of hepatic histological improvement in chronic hepatitis C patients responding to interferon treatment.

BACKGROUND AND METHODS: Serum total bile acid (TBA) levels are used clinically as a sensitive and reliable index of hepatobiliary diseases. In the present study, to assess the clinical usefulness of determining TBA in interferon (IFN)-treated patients, changes in liver function test values, including TBA and liver histology, were examined in 36 chronic hepatitis C patients for 3 years after a sustained response to IFN treatment. RESULTS: Alanine aminotransferase and gamma-glutamyl transpeptidase values significantly decreased during the period of IFN treatment compared to the same measures before IFN treatment. Albumin, cholinesterase, total cholesterol and platelet count values temporarily decreased during IFN treatment, then increased significantly and reached a plateau 6-12 months after the end of IFN treatment. The zinc sulphate turbidity test and TBA values began to decrease during IFN treatment and continued to decrease during the 3-year follow-up period. The histological activity index of the liver (Knodell's score) significantly decreased, whereas the staging score was unchanged 1 year after the end of IFN treatment. In patients who had a TBA value > 10 micromol/L before IFN treatment, a significant correlation was observed between the decrease of TBA and liver histology grading score. CONCLUSIONS: A decrease in serum TBA level reflected histological improvement in the liver more precisely than did changes in the other liver function test values following IFN therapy.

Stromal cell-independent differentiation of human cord blood CD34+CD38- lymphohematopoietic progenitors toward B cell lineage.

To study the cytokine regulation of early stages of human B-lymphopoiesis, we developed a stroma-free two-step culture system. Single human cord blood CD34+CD38- cells were individually cultured by micromanipulation with interleukin (IL)-3, stem cell factor (SCF), fIt3 ligand (FL), IL-6 and granulocyte colony-stimulating factor (G-CSF). About 10% of the cells formed primary colonies, which were individually tested for myeloid and B-lymphoid potentials by reculturing aliquots of the primary colony cells into secondary myeloid and B-lymphoid cultures. One third of the primary colonies proved capable of differentiation into CD19+IgM+ cells, as well as into myeloid lineage cells. RT-PCR analyses revealed that some cells in the primary culture had already matured to express B cell-specific transcripts. Thus, the combination of IL-3, SCF, FL, IL-6 and G-CSF supported the differentiation of CD34+CD38- lymphohematopoietic progenitors toward B cell lineage in addition to myeloid lineages. Screening of cytokines to identify the minimum requirement of cytokines in the primary culture revealed that IL-3 and SCF were essential and that the addition of FL, and to a lesser extent IL-6 or G-CSF, to the combination of IL3 and SCF remarkably enhanced the primary colony formation and the generation of CD19+ cells in the secondary B-lymphoid culture.

Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein.

alpha-Synuclein has been implicated in the pathogenesis of Parkinson's disease, since rare autosomal dominant mutations are associated with early onset of the disease and alpha-synuclein was found to be a major constituent of Lewy bodies. We have analyzed alpha-synuclein expression in transfected cell lines. In pulse-chase experiments alpha-synuclein appeared to be stable over long periods (t((1)/(2)) 54 h) and no endoproteolytic processing was observed. alpha-Synuclein was constitutively phosphorylated in human kidney 293 cells as well as in rat pheochromocytoma PC12 cells. In both cell lines phosphorylation was highly sensitive to phosphatases, since okadaic acid markedly stabilized phosphate incorporation. Phosphoamino acid analysis revealed that phosphorylation occurred predominantly on serine. Using site-directed mutagenesis we have identified a major phosphorylation site at serine 129 within the C-terminal domain of alpha-synuclein. An additional site, which was phosphorylated less efficiently, was mapped to serine 87. The major phosphorylation site was located within a consensus recognition sequence of casein kinase 1 (CK-1). In vitro experiments and two-dimensional phosphopeptide mapping provided further evidence that serine 129 was phosphorylated by CK-1 and CK-2. Moreover, phosphorylation of serine 129 was reduced in vivo upon inhibition of CK-1 or CK-2. These data demonstrate that alpha-synuclein is constitutively phosphorylated within its C terminus and may indicate that the function of alpha-synuclein is regulated by phosphorylation/dephosphorylation.

Cellular distribution of the splice variants of the receptor for pituitary adenylate cyclase-activating polypeptide (PAC(1)-R) in the rat brain by in situ RT-PCR.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a pleiotropic neuropeptide and its specific receptor (the PAC(1) receptor) is widely distributed in the rat brain. It has been reported that alternative splicing of the region encoding the third intracellular loop of the PAC(1) receptor generates six isoforms which are differentially coupled to signal transduction pathways, but the precise distribution and localization of these splice isoforms in the brain remain to be determined. Using the initial specific primer pairs which correspond to the 'hip' or 'hop' types of receptors for the solution-phase reverse transcription-polymerase chain reaction (RT-PCR), we demonstrated that the major splice variants of the PAC(1) receptor in various regions of the rat brain are the short splice isoform 'PAC(1)-R-s' which does not contain either the 'hip' or 'hop' cassette and the another splice isoform, 'PAC(1)-R-hop', which contains the 'hop' cassette. With an innovative molecular histochemical technique, in situ RT-PCR, we determined that these two splice isoforms are both intensely expressed in the mitral cells of the olfactory bulb, the Purkinje cells of the cerebellum, the pyramidal cells of the hippocampus and neocortex, and many neurons in the nuclei of hypothalamus and thalamus as well as other regions. The initial mapping of the cell type-specific expression of these two splice variants of the PAC(1) receptor provides the basis for a better understanding of the functional significance of the PAC(1)-R and its ligand PACAP in various brain regions.

Postmortem absorption of dichloromethane: a case study and animal experiments.

A case of accidental death after occupational exposure to an atmosphere containing dichloromethane (DCM) is reported. The concentrations of DCM in the blood and tissues of a 40-year-old man who died while observing an industrial washing machine filled with DCM vapour were blood 1660 mg/l, urine 247 mg/l, brain 87 mg/kg, heart muscle 199 mg/kg and lungs 103 mg/kg which are 3-7 times higher than previously reported fatal levels. The body was left undiscovered in the machine filled with DCM vapour for about 20 h. The present study was designed to determine whether all the DCM detected in the tissues and body fluids had been inhaled while alive using rats as the experimental model. The concentrations of DCM in the tissues and body fluids of a rat that died from DCM poisoning and was left for 20 h in a box containing DCM vapour were the same as those in the tissues and body fluids of a rat that had died from an injected overdose of barbiturates and had then been placed in the DCM box in a similar manner. Moreover, the concentrations of DCM in the tissues and body fluids of the carcasses that were exposed to the DCM vapour increased gradually throughout the period of exposure. These findings imply that DCM is able to penetrate the tissues and body fluids of rat carcasses through a route other than inhalation such as through the skin.