Development of an Anti-Adhesive Membrane for Use in Video-Assisted Thoracic Surgery.

Background: The need to prevent postoperative adhesions after surgery has been considered a significant challenge in thoracic surgery, especially with the advent of video-assisted thoracic surgery (VATS). While preventive materials for postoperative adhesions have been studied for many years, they are all still in the development phases. Methods: In this animal study, an insoluble hyaluronic acid membrane was used in VATS for wedge resection to test its operability and to examine the body's response to the membrane. Ten beagles were divided into two groups, an experimental group and a negative control group. In the experimental group, an insoluble hyaluronic acid membrane containing glycerol was used as the test membrane (10 x 10 x 0.1 cm3). The test membrane was implanted in the left thoracic cavity of the animal under VATS following wedge resection. The animals were observed for two weeks and then euthanized for examination. Results: Macroscopically, the median adhesion score was lower in the experimental group (0) than in the control group (2.5). On histopathological examination, the test membrane elicited only a minor inflammatory response and foreign body reaction. Conclusion: The test membrane showed satisfactory operability and appears to be a practical material to prevent postoperative adhesions after thoracic surgery in VATS.

Downregulation of TMEM70 in Rat Liver Cells After Hepatocarcinogen Treatment Related to the Warburg Effect in Hepatocarcinogenesis Producing GST-P-Expressing Proliferative Lesions.

We previously observed downregulation of mitochondrial oxidative phosphorylation-related protein, TMEM70, which is suggestive of disrupted cellular senescence, in GST-P-expressing (+) proliferative lesions from early hepatocarcinogenesis stages in rats. The present study investigated the immunohistochemical relationship between TMEM70 downregulation and cellular metabolic changes in carcinogenic processes, as well as the onset of the liver cell respiratory changes after repeated hepatocarcinogen treatment in rats. At the early hepatocarcinogenesis stage in a 2-stage model, GST-P+ preneoplastic lesions showing TMEM70 downregulation also downregulated the mitochondrial ATPase, ATPB, but upregulated glycolysis-related glucose transporter member 1 (GLUT1) and glucose-6-phosphate dehydrogenase, suggesting a metabolic shift from oxidative phosphorylation to glycolysis, known as the Warburg effect. Combined downregulation of TMEM70 and ATPB increased proliferation activity in GST-P+ preneoplastic lesions, suggesting cell proliferation facilitation by reducing mitochondrial respiration. Concurrent GLUT1-upregulation and TMEM70-downregulation increased nuclear phosphorylated c-MYC+ cells in GST-P+ preneoplastic lesions, suggesting c-MYC-mediated transcription facilitation to promote glycolysis and cell proliferation. The TMEM70-related metabolic shift was enhanced in GST-P+ neoplastic lesions, suggesting a contribution to tumor progression. Conversely, the TMEM70-related metabolic shift was lacking in peroxisome proliferator-activated receptor-α agonist-induced hepatocarcinogenesis, as well as in carcinogenic processes targeting other organs. Transcript expression analysis following 28- and 90-day repeated hepatocarcinogen treatment showed downregulation of Tmem70 from day 28 and upregulation of Pkm and Myc at day 90, suggesting early onset of a catastrophic cellular senescence-related metabolic shift beginning from depressed mitochondrial respiration in the liver. These results suggest a contribution of TMEM70 downregulation to the Warburg effect, which directs tumor promotion and progression in GST-P+-linked hepatocarcinogenesis in rats.

Japan Flavour and Fragrance Materials Association's (JFFMA) safety assessment of food-flavouring substances uniquely used in Japan that belong to the class of aliphatic primary alcohols, aldehydes, carboxylic acids, acetals and esters containing additional oxygenated functional groups.

We performed a safety evaluation using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) of the following four flavouring substances that belong to the class of 'aliphatic primary alcohols, aldehydes, carboxylic acids, acetals, and esters containing additional oxygenated functional groups' and are uniquely used in Japan: butyl butyrylacetate, ethyl 2-hydroxy-4-methylpentanoate, 3-hydroxyhexanoic acid and methyl hydroxyacetate. Although no genotoxicity study data were found in the published literature, none of the four substances had chemical structural alerts predicting genotoxicity. All four substances were categorised as class I by using Cramer's classification. The estimated daily intake of each of the four substances was determined to be 0.007-2.9 µg/person/day by using the maximised survey-derived intake method and based on the annual production data in Japan in 2001, 2005 and 2010, and was determined to be 0.250-600.0 µg/person/day by using the single-portion exposure technique and based on average-use levels in standard portion sizes of flavoured foods. Both of these estimated daily intake ranges were below the threshold of toxicological concern for class I substances, which is 1800 µg/person/day. Although no information from in vitro and in vivo toxicity studies for the four substances was available, these substances were judged to raise no safety concerns at the current levels of intake.

Late Effect of Developmental Exposure to 3,3'-Iminodipropionitrile on Neurogenesis in the Hippocampal Dentate Gyrus of Mice.

The effects of developmental exposure to 3,3'-iminodipropionitrile (IDPN), a neurotoxicant that causes proximal axonopathy, on mouse hippocampal neurogenesis was examined. Pregnant mice were exposed to IDPN at 0, 600, or 1200 ppm in their drinking water from gestational day 6 to postnatal day (PND) 21. On PND 21, male offspring showed increased postmitotic neuron-specific NeuN-immunoreactive(+) granule cell numbers in the dentate subgranular zone (SGZ) and granule cell layer (GCL) and decreased glutamate receptor gene Grin2d levels in the dentate gyrus at 1200 ppm. On PND 77, decreased numbers were observed for TBR2+ progenitor cells in the SGZ at ≥600 ppm and GFAP+ stem cells, DCX+ progenitor cells and immature granule cells, NeuN+ immature and mature granule cells, PCNA+ proliferating cells in the SGZ and/or GCL, and immunoreactive cells for ARC or FOS, immediate-early gene products related to neuronal and synaptic plasticity, in the GCL at 1200 ppm. Additionally, at 1200 ppm of IDPN, downregulation of Kit, the gene encoding the stem cell factor (SCF) receptor, and upregulation of Kitl, encoding SCF, were observed in the dentate gyrus. Therefore, maternal IDPN exposure in mice affects neurogenesis involving glutamatergic signals at the end of developmental exposure, with late effects suppressing SGZ cell proliferation, reducing the broad range of granule cell lineage population, which may be responsible for SCF receptor downregulation. The upregulated SCF was likely a feedback response to the decreased receptor level. These results suggest that reduced SCF signaling may cause suppressed neuronal and synaptic plasticity.

Maternal Exposure to Valproic Acid Primarily Targets Interneurons Followed by Late Effects on Neurogenesis in the Hippocampal Dentate Gyrus in Rat Offspring.

Valproic acid (VPA) is used to establish models of experimental autism. The present study investigated the developmental exposure effect of VPA on postnatal hippocampal neurogenesis in accordance with the exposure scheme of OECD Test Guideline 426 adopted for developmental neurotoxicity. Pregnant rats were administered drinking water containing 0, 667, or 2000 ppm VPA from gestational day 6 until day 21 post-delivery. In the subgranular zone (SGZ) and granule cell layer (GCL) of offspring, the number of granule cell lineage subpopulations remained unchanged upon weaning. However, in the hilus of the dentate gyrus, the number of reelin+ interneurons decreased at ≥667 ppm, and the number of PVALB+ or GAD67+ interneurons decreased at 2000 ppm. Conversely, Reln and Gad1 transcript levels increased at 2000 ppm, but Pvalb and Grin2d decreased, in the dentate gyrus. At the adult stage, PCNA+ proliferating SGZ cells, NeuN+ postmitotic SGZ/GCL neurons, and ARC+ or COX2+ GCL neurons increased at ≥667 ppm. In the dentate hilus, decreases in GAD67+ interneuron subpopulations and Grin2d transcript levels sustained at 2000 ppm. These results suggested that VPA primarily targets interneurons by developmental exposure, and this is followed by late effects on granule cell lineages, likely by influencing SGZ cell proliferation and synaptic plasticity. A reduced population of reelin+ or PVALB+ interneurons did not affect distribution of granule cell lineage subpopulations upon weaning. The late effect on neurogenesis, which resulted in increased GCL neurons, might be the result of a sustained decrease in GAD67+ interneurons expressing NR2D encoded by Grin2d.

Maternal exposure to ochratoxin A targets intermediate progenitor cells of hippocampal neurogenesis in rat offspring via cholinergic signal downregulation and oxidative stress responses.

To elucidate the developmental exposure effects of ochratoxin A (OTA) on postnatal hippocampal neurogenesis, pregnant SD rats were provided a diet containing 0, 0.12, 0.6, or 3.0ppm OTA from gestation day 6 to day 21 on weaning after delivery. Offspring were maintained through postnatal day (PND) 77 without OTA exposure. At 3.0ppm, offspring of both sexes showed a transient body weight decrease after weaning. Changes in hippocampal neurogenesis-related parameters as measured in male PND 21 offspring were observed at 3.0ppm. In the subgranular zone (SGZ) of the dentate gyrus, PAX6+ or TBR2+ cells were decreased, while GFAP+ or DCX+ cells did not fluctuate in number, suggesting decreased numbers of type-2 progenitor cells. On the other hand, SGZ cells accumulating malondialdehyde increased. In the hilus of the dentate gyrus, SST+ or CHRNB2+ γ-aminobutyric acid (GABA)-ergic interneurons decreased, accompanied with transcript downregulation of Chrnb2 in the dentate gyrus. These results suggest that maternal exposure to OTA decreased type-2 progenitor cells by reducing hilar GABAergic interneurons innervating type-2 progenitor cells via cholinergic signal downregulation and also by increasing oxidative stress in the SGZ. Transcript levels of neurotrophin (Bdnf), glutamatergic receptors (Gria1, Gria2, and Grin2a), serotonin-synthesizing enzyme, and serotonergic receptors (Tph2, Htr1a, and Htr4) increased in the dentate gyrus, suggesting multiple neuroprotective actions against OTA-induced aberrant neurogenesis. All observed fluctuations were reversed by PND 77. The no-observed-adverse-effect level for offspring neurogenesis was determined to be 0.6ppm, corresponding to 39.3-76.0µg/kg body weight/day.

Disruption of spindle checkpoint function in rats following 28 days of repeated administration of renal carcinogens.

We previously reported that 28-day exposure to hepatocarcinogens that facilitate cell proliferation specifically alters the expression of G1/S checkpoint-related genes and proteins, induces aberrant early expression of ubiquitin D (UBD) at the G2 phase, and increases apoptosis in the rat liver, indicating G1/S and spindle checkpoint dysfunction. The present study aimed to determine the time of onset of carcinogen-specific cell-cycle disruption after repeated administration of renal carcinogens for up to 28 days. Rats were orally administered the renal carcinogens nitrofurantoin (NFT), 1-amino-2,4-dibromoantraquinone (ADAQ), and 1,2,3-trichloropropane (TCP) or the non-carcinogenic renal toxicants 1-chloro-2-propanol, triamterene, and carboxin for 3, 7 or 28 days. Both immunohistochemical single-molecule analysis and real-time RT-PCR analysis revealed that carcinogen-specific expression changes were not observed after 28 days of administration. However, the renal carcinogens ADAQ and TCP specifically reduced the number of cells expressing phosphorylated-histone H3 at Ser10 in both UBD(+) cells and proliferating cells, suggestive of insufficient UBD expression at the M phase and early transition of proliferating cells from the M phase, without increasing apoptosis, after 28 days of administration. In contrast, NFT, which has marginal carcinogenic potential, did not induce such cellular responses. These results suggest that it may take 28 days to induce spindle checkpoint dysfunction by renal carcinogens; however, induction of apoptosis may not be essential. Thus, induction of spindle checkpoint dysfunction may be dependent on carcinogenic potential of carcinogen examined, and marginal carcinogens may not exert sufficient responses even after 28 days of administration.

Developmental cuprizone exposure impairs oligodendrocyte lineages differentially in cortical and white matter tissues and suppresses glutamatergic neurogenesis signals and synaptic plasticity in the hippocampal dentate gyrus of rats.

Developmental cuprizone (CPZ) exposure impairs rat hippocampal neurogenesis. Here, we captured the developmental neurotoxicity profile of CPZ using a region-specific expression microarray analysis in the hippocampal dentate gyrus, corpus callosum, cerebral cortex and cerebellar vermis of rat offspring exposed to 0, 0.1, or 0.4% CPZ in the maternal diet from gestation day 6 to postnatal day (PND) 21. Transcripts of those genes identified as altered were subjected to immunohistochemical analysis on PNDs 21 and 77. Our results showed that transcripts for myelinogenesis-related genes, including Cnp, were selectively downregulated in the cerebral cortex by CPZ at ≥0.1% or 0.4% on PND 21. CPZ at 0.4% decreased immunostaining intensity for 2',3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase) and CNPase(+) and OLIG2(+) oligodendrocyte densities in the cerebral cortex, whereas CNPase immunostaining intensity alone was decreased in the corpus callosum. By contrast, a striking transcript upregulation for Klotho gene and an increased density of Klotho(+) oligodendrocytes were detected in the corpus callosum at ≥0.1%. In the dentate gyrus, CPZ at ≥0.1% or 0.4% decreased the transcript levels for Gria1, Grin2a and Ptgs2, genes related to the synapse and synaptic transmission, and the number of GRIA1(+) and GRIN2A(+) hilar γ-aminobutyric acid (GABA)-ergic interneurons and cyclooxygenase-2(+) granule cells. All changes were reversed at PND 77. Thus, developmental CPZ exposure reversibly decreased mature oligodendrocytes in both cortical and white matter tissues, and Klotho protected white matter oligodendrocyte growth. CPZ also reversibly targeted glutamatergic signals of GABAergic interneuron to affect dentate gyrus neurogenesis and synaptic plasticity in granule cells.

Disruption of spindle checkpoint function ahead of facilitation of cell proliferation by repeated administration of hepatocarcinogens in rats.

We aimed to clarify the hepatocarcinogen-specific disruption of cell cycle checkpoint functions and its time course after repeated administration of hepatocarcinogens. Thus, rats were repeatedly administered with hepatocarcinogens (methapyrilene, carbadox and thioacetamide), a marginal hepatocarcinogen (leucomalachite green), hepatocarcinogenic promoters (oxfendazole and ß-naphthoflavone) or non-carcinogenic hepatotoxicants (promethazine and acetaminophen) for 7, 28 or 90 days, and the temporal changes in cell proliferation, expression of G1/S and spindle checkpoint-related molecules, and apoptosis were examined using immunohistochemistry and/or real-time RT-PCR analysis. Hepatocarcinogens facilitating cell proliferation at day 28 of administration also facilitated cell proliferation and apoptosis at day 90. Hepatocarcinogen- or hepatocarcinogenic promoter-specific cellular responses were not detected by immunohistochemical single molecule analysis even after 90 days. Expression of Cdkn1a, Mad2l1, Chek1 and Rbl2 mRNA also lacked specificity to hepatocarcinogens or hepatocarcinogenic promoters. In contrast, all hepatocarcinogens and the marginally hepatocarcinogenic leucomalachite green induced Mdm2 upregulation or increase in the number of phosphorylated MDM2(+) cells from day 28, irrespective of the lack of cell proliferation facilitation by some compounds. However, different Tp53 expression levels suggest different mechanisms of induction or activation of MDM2 among hepatocarcinogens. On the other hand, hepatocarcinogenic methapyrilene and carbadox downregulated the number of both ubiquitin D(+) cells and proliferating cells remaining in M phase at day 28 and/or day 90, irrespective of the lack of cell proliferation facilitation in the latter. These results suggest that hepatocarcinogens disrupt spindle checkpoint function after 28 or 90 days of administration, which may be induced ahead of cell proliferation facilitation.

Developmental exposure of aflatoxin B1 reversibly affects hippocampal neurogenesis targeting late-stage neural progenitor cells through suppression of cholinergic signaling in rats.

To elucidate the maternal exposure effects of aflatoxin B1 (AFB1) and its metabolite aflatoxin M1, which is transferred into milk, on postnatal hippocampal neurogenesis, pregnant Sprague-Dawley rats were provided a diet containing AFB1 at 0, 0.1, 0.3, or 1.0 ppm from gestational day 6 to day 21 after delivery on weaning. Offspring were maintained through postnatal day (PND) 77 without AFB1 exposure. Following exposure to 1.0 ppm AFB1, offspring showed no apparent systemic toxicity at weaning, whereas dams showed increased liver weight and DNA repair gene upregulation in the liver. In the hippocampal dentate gyrus of male PND 21 offspring, the number of doublecortin(+) progenitor cells were decreased, which was associated with decreased proliferative cell population in the subgranular zone at ≥ 0.3 ppm, although T-box brain 2(+) cells, tubulin beta III(+) cells, gamma-H2A histone family, member X(+) cells, and cyclin-dependent kinase inhibitor 1A(+) cells did not fluctuate in number. AFB1 exposure examined at 1.0 ppm also resulted in transcript downregulation of the cholinergic receptor subunit Chrna7 and dopaminergic receptor Drd2 in the dentate gyrus, although there was no change in transcript levels of DNA repair genes. In the hippocampal dentate hilus, interneurons expressing CHRNA7 or phosphorylated tropomyosin receptor kinase B (TRKB) decreased at ≥ 0.3 ppm. On PND 77, there were no changes in neurogenesis-related parameters. These results suggested that maternal AFB1 exposure reversibly affects hippocampal neurogenesis targeting type-3 progenitor cells. This mechanism likely involves suppression of cholinergic signals on hilar GABAergic interneurons and brain-derived neurotrophic factor-TRKB signaling from granule cells. The no-observed-adverse-effect level for offspring neurogenesis was determined to be 0.1 ppm (7.1-13.6 mg/kg body weight/day).

The Japan Flavour and Fragrance Materials Association's (JFFMA) safety assessment of acetal food flavouring substances uniquely used in Japan.

Using the procedure devised by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), we performed safety evaluations on five acetal flavouring substances uniquely used in Japan: acetaldehyde 2,3-butanediol acetal, acetoin dimethyl acetal, hexanal dibutyl acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal. As no genotoxicity study data were available in the literature, all five substances had no chemical structural alerts predicting genotoxicity. Using Cramer's classification, acetoin dimethyl acetal and hexanal dibutyl acetal were categorised as class I, and acetaldehyde 2,3-butanediol acetal, hexanal glyceryl acetal and 4-methyl-2-pentanone propyleneglycol acetal as class III. The estimated daily intakes for all five substances were within the range of 1.45-6.53 µg/person/day using the method of maximised survey-derived intake based on the annual production data in Japan from 2001, 2005, 2008 and 2010, and 156-720 µg/person/day using the single-portion exposure technique (SPET), based on the average use levels in standard portion sizes of flavoured foods. The daily intakes of the two class I substances were below the threshold of toxicological concern (TTC) - 1800 µg/person/day. The daily intakes of the three class III substances exceeded the TTC (90 µg/person/day). Two of these, acetaldehyde 2,3-butanediol acetal and hexanal glyceryl acetal, were expected to be metabolised into endogenous products after ingestion. For 4-methyl-2-pentanone propyleneglycol acetal, one of its metabolites was not expected to be metabolised into endogenous products. However, its daily intake level, based on the estimated intake calculated by the SPET method, was about 1/15 000th of the no observed effect level. It was thus concluded that all five substances raised no safety concerns when used for flavouring foods at the currently estimated intake levels. While no information on in vitro and in vivo toxicity for all five substances was available, their metabolites were judged as raising no safety concerns at the current levels of intake.

Intratracheal instillation methods and the distribution of administered material in the lung of the rat.

Intratracheal instillation is widely used for respiratory toxicity tests in experimental animals. However, there are wide variations in the techniques used for instillation, and it is thus difficult to compare the results obtained using different techniques. To examine the effect of instillation methods, we compared the distribution of a test substance in the lungs of rats after intratracheal instillations under various conditions. Rats received an intratracheal instillation of 0.3 mL of india ink suspension under different conditions as follows: 3 different angles of body restraint, 0° (supine horizontal), 45° (supine head up) and 90° (vertical head up); 2 instillation speeds, high (40 mL/min) and low (4 mL/min); and 2 different devices, a standard bulb-tipped gavage needle and an aerosolizing microsprayer designed for intratracheal instillation. One hour after treatment under these various conditions, rats were sacrificed, and the local distribution of the suspension in the lungs was observed. No animal restrained in the supine head-up or vertical head-up position died from the treatment; however, fatalities were observed when rats were restrained in the supine horizontal position except under high-speed dosing conditions with a microsprayer. Better distribution of the suspension in the lungs was observed in the rats restrained in the supine head-up position after instillation at high speed when compared with other conditions. These results indicated that high-speed instillation to the subject restrained in the supine head-up position is an appropriate condition for performing intratracheal instillation.

Endocrine disrupting effects of low dose 17 ß-estradiol (E2) on the Japanese quail (Coturnix japonica) were detected by modified one-generation reproduction study.

Previously, we investigated endocrine disrupting effects of 17 ß-estradiol (E(2)) on Japanese quail (Coturnix japonica) in the avian reproduction test according to the testing guidelines, in which new endpoints such as blood vitellogenin (VTG) concentration in parent quails and pathology of F(1) chicks were added, and consequently these additional endpoints suggested to be sensitive markers for detecting any impacts of endocrine disrupting effects (Shibuya et al., 2005b). In the present study, to investigate low dose effects of estrogenic endocrine disrupting chemicals in birds, the avian reproduction study of E(2) at low dose levels was conducted using Japanese quail with additional endpoints such as observations of F(1) chicks until 10 weeks of age, histopathology of F(1) chicks at 14 days and 10 weeks of age and blood VTG concentration in parent quails. Sixteen pairs of 10-week-old quails were fed a low phytoestrogen diet containing E(2) at 0 (control), 0.3, 3, and 30 ppm for 6 weeks, and parent quails, eggs and offspring were examined. F(1) chicks were maintained up to 14 days or 10 weeks of age. Serum E(2) and VTG concentrations in males of the E(2) 3- and 30-ppm groups and in females of the E(2) 30-ppm groups were significantly elevated. In the E(2) 30-ppm group, two parent females died, and toxic changes such as suppression of body weight gain, decrease in food consumption and atrophic and degenerative changes of the reproductive organs were observed in parent quails. In the same group, the number of eggs laid and the fertility rate of eggs were significantly decreased. In addition, the viability of F(1) chicks in the E(2) 30-ppm group were significantly decreased at 10 weeks of age. On the other hand, no abnormalities described above were observed in any parent quails, eggs and F(1) chicks in the E(2) 3- and 0.3-ppm groups, although the fertility rates of eggs in both groups were decreased and the body weight gain of F(1) females in the E(2) 3-ppm group was significantly suppressed. In the histopathological examination of F(1) chicks maintained up to 10 weeks of age, persistent right oviduct and atrophy of the oviduct gland were observed in females of E(2)-treatment groups with significantly high incidences. Moreover, cystic dilatation and tubular degeneration of the seminiferous tubules and atrophy of the cloacal gland were also observed in males of the E(2)-treatment groups. Thus, the dietary treatment of low dose E(2) (even 0.3 ppm) to parent quails resulted in decreased viability and induction of abnormalities in the oviduct, testis and cloacal gland in F(1) chicks maintained up to 10 weeks of age. These results suggest that additional endpoints such as observations of F(1) chicks until 10 weeks of age, histopathology of F(1) chicks at 14 days and 10 weeks of age and blood VTG concentration in parent quails would be useful and sensitive endpoints for evaluating estrogenic endocrine disrupting effects in the avian reproduction study.

Changes in TIMP-1 and -2 expression in the early stage of porcine serum-induced liver fibrosis in rats.

It is widely recognized that tissue inhibitors of metalloproteinases (TIMPs), especially TIMP-1 and -2, play a key role in the progression of hepatic fibrosis. In the present study, we examined the changes in TIMP-1 and -2 expressions in the early stage of porcine serum (PS)-induced liver fibrosis in Brown Norway (BN) and Wistar rats. The rats were injected intraperitoneally with 0.5 ml/head of PS twice a week for up to 8 weeks and examined at 2, 4 and 8 weeks. Hepatic fibrosis and inflammatory cell infiltration developed at 4 and 8 weeks in BN and Wistar rats, respectively, and formation of pseudolobules was detected at 8 weeks in rats of both strains. The expression of liver TIMP-1 and -2 mRNAs significantly increased at 8 weeks in rats of both strains. At the same time, TIMP-1 and -2 activities were also detected in the liver of both strains. On the other hand, the expression of serum TIMP-1 and -2 proteins increased earlier (at 4 weeks for TIMP-1 and at 2 or 4 weeks for TIMP-2) than that of liver TIMP-1 and -2 mRNAs did. Although there are some reports suggestive of why the elevation of serum TIMP-1 and -2 proteins preceded that of liver TIMP-1 and -2 mRNAs, the exact reason is still obscure. In conclusion, the present study showed for the first time the mode of TIMP-1 and -2 expression and activity in the early stage of PS-induced rat liver fibrosis model.