Therapeutic potential of hepatocyte-like-cells converted from stem cells from human exfoliated deciduous teeth in fulminant Wilson's disease.

Wilson's disease (WD) is an inherited metabolic disease arising from ATPase copper transporting beta gene (ATP7B) mutation. Orthotoropic liver transplantation is the only radical treatment of fulminant WD, although appropriate donors are lacking at the onset of emergency. Given the hepatogenic capacity and tissue-integration/reconstruction ability in the liver of stem cells from human exfoliated deciduous teeth (SHED), SHED have been proposed as a source for curing liver diseases. We hypothesized the therapeutic potential of SHED and SHED-converted hepatocyte-like- cells (SHED-Heps) for fulminant WD. SHED and SHED-Heps were transplanted into WD model Atp7b-mutated Long-Evans Cinnamon (LEC) rats received copper overloading to induce a lethal fulminant liver failure. Due to the superior copper tolerance via ATP7B, SHED-Hep transplantation gave more prolonged life-span of fulminant LEC rats than SHED transplantation. The integrated ATP7B-expressing SHED-Heps showed more therapeutic effects on to restoring the hepatic dysfunction and tissue damages in the recipient liver than the integrated naïve SHED without ATP7B expression. Moreover, SHED-Heps could reduce copper-induced oxidative stress via ATP7B- independent stanniocalcin 1 secretion in the fulminant LEC rats, suggesting a possible role for paracrine effect of the integrated SHED-Heps. Taken together, SHED-Heps offer a potential of functional restoring, bridging, and preventive approaches for treating fulminant WD.

Demonstrating Potential of Cell Therapy for Wilson's Disease with the Long-Evans Cinnamon Rat Model.

Wilson's disease (WD) is characterized by the inability to excrete copper (Cu) from the body with progressive tissue injury, especially in liver and brain. The molecular defect in WD concerns mutations in ATP7B gene leading to loss of Cu transport from the hepatocyte to the bile canaliculus. While drugs, e.g., Cu chelators, have been available for several decades, these must be taken lifelong, which can be difficult due to issues of compliance or side effects. Many individuals may require liver transplantation, which can also be difficult due to donor organ shortages. Therefore, achieving permanent cures via cell or gene therapy are of great interest for WD. Cell therapy is feasible because transplanted hepatocytes can integrate in liver parenchyma and restore deficient functions, including transport of Cu into bile. The availability of authentic animal models that recapitulate hepatic WD, especially the Long-Evans Cinnamon (LEC) rat, has advanced cell transplantation research in WD. We describe requirements for cell therapy in animal models with several standardized methods for studies to test or refine cell therapy strategies in WD.

Nonclinical and pharmacokinetic assessments to evaluate the potential of tedizolid and linezolid to affect mitochondrial function.

Prolonged treatment with the oxazolidinone linezolid is associated with myelosuppression, lactic acidosis, and neuropathies, toxicities likely caused by impairment of mitochondrial protein synthesis (MPS). To evaluate the potential of the novel oxazolidinone tedizolid to cause similar side effects, nonclinical and pharmacokinetic assessments were conducted. In isolated rat heart mitochondria, tedizolid inhibited MPS more potently than did linezolid (average [± standard error of the mean] 50% inhibitory concentration [IC50] for MPS of 0.31 ± 0.02 µM versus 6.4 ± 1.2 µM). However, a rigorous 9-month rat study comparing placebo and high-dose tedizolid (resulting in steady-state area under the plasma concentration-time curve values about 8-fold greater than those with the standard therapeutic dose in humans) showed no evidence of neuropathy. Additional studies explored why prolonged, high-dose tedizolid did not cause these mitochondriopathic side effects despite potent MPS inhibition by tedizolid. Murine macrophage (J774) cell fractionation studies found no evidence of a stable association of tedizolid with eukaryotic mitochondria. Monte Carlo simulations based on population pharmacokinetic models showed that over the course of a dosing interval using standard therapeutic doses, free plasma concentrations fell below the respective MPS IC50 in 84% of tedizolid-treated patients (for a median duration of 7.94 h) and 38% of linezolid-treated patients (for a median duration of 0 h). Therapeutic doses of tedizolid, but not linezolid, may therefore allow for mitochondrial recovery during antibacterial therapy. The overall results suggest that tedizolid has less potential to cause myelosuppression and neuropathy than that of linezolid during prolonged treatment courses. This, however, remains a hypothesis that must be confirmed in clinical studies.

Anti-inflammatory activity of grains of paradise (Aframomum melegueta Schum) extract.

The ethanolic extract of grains of paradise (Aframomum melegueta Schum, Zingiberaceae) has been evaluated for inhibitory activity on cyclooxygenase-2 (COX-2) enzyme, in vivo for the anti-inflammatory activity and expression of several pro-inflammatory genes. Bioactivity-guided fractionation showed that the most active COX-2 inhibitory compound in the extract was [6]-paradol. [6]-Shogaol, another compound from the extract, was the most active inhibitory compound in pro-inflammatory gene expression assays. In a rat paw edema model, the whole extract reduced inflammation by 49% at 1000 mg/kg. Major gingerols from the extract [6]-paradol, [6]-gingerol, and [6]-shogaol reduced inflammation by 20, 25 and 38%. respectively when administered individually at a dose of 150 mg/kg. [6]-Shogaol efficacy was at the level of aspirin, used as a positive control. Grains of paradise extract has demonstrated an anti-inflammatory activity, which is in part due to the inhibition of COX-2 enzyme activity and expression of pro-inflammatory genes.

Coffee consumption delays the hepatitis and suppresses the inflammation related gene expression in the Long-Evans Cinnamon rat.

BACKGROUND AND AIMS: Large-scale epidemiological studies have shown that drinking more than two cups of coffee per day reduces the risks of hepatitis and liver cancer. However, the heterogeneity of the human genome requires studies of experimental animal models with defined genetic backgrounds to evaluate the coffee effects on liver diseases. We evaluated the efficacy of coffee consumption with one of experimental animal models for human disease. METHOD: We used the Long Evans Cinnamon (LEC) rat, which onsets severe hepatitis and high incidence of liver cancer, due to the accumulation of copper and iron in livers caused by the genetic mutation in Atp7B gene, and leading to the continuous oxidative stress. We determined the expression of inflammation related genes, and amounts of copper and iron in livers, and incidence of the pre-neoplastic foci in the liver tissue of LEC rats. RESULTS: Coffee administration for 25 weeks delayed the occurrence of hepatitis by two weeks, significantly improved survival, reduced the expression of inflammatory cytokines, and reduced the incidence of small pre-neoplastic liver foci in LEC rats. There was no significant difference in the accumulation of copper and iron in livers, indicating that coffee administration does not affect to the metabolism of these metals. These findings indicate that drinking coffee potentially prevents hepatitis and liver carcinogenesis through its anti-inflammatory effects. CONCLUSION: This study showed the efficacy of coffee in the prevention of hepatitis and liver carcinogenesis in the LEC model.

Developmental timing of the effects of maternal care on gene expression and epigenetic regulation of hormone receptor levels in female rats.

Maternal care experienced during postnatal development has enduring effects on neuroendocrine function and behavior. Previous studies in rats have illustrated the effect of maternal licking/grooming (LG) on hormone receptors and maternal behavior of adult female offspring associated with altered DNA methylation. However, the developmental timing of these effects, which provide insight into the cellular and molecular pathways through which early experience alters later behavior, had not been explored. Here, we demonstrate the developmental emergence of these outcomes and use cross-fostering to identify sensitive periods for these effects. Estrogen receptor (ER)α and ERß mRNA levels within the medial preoptic area (MPOA) of the hypothalamus were increased by postnatal day (PN)21 in female offspring of high LG dams; LG-associated increases in oxytocin receptor mRNA levels were observed beyond the weaning period. Quantification of ERα-immunoreactivity indicated a high degree of neuroanatomical specificity of LG effects within the MPOA that were observed by PN6. Reduced DNA methylation and histone 3 lysine 9 tri-methylation and increased histone 3 lysine 4 tri-methylation at the ERα gene promoter (Esr1) were detected at PN21 in high LG female offspring. Latency to engage in maternal behavior toward donor pups was significantly shorter among high LG females. Cross-fostering revealed that maternal sensitization and MPOA ERα levels are sensitive to maternal care experienced before but not after PN10. Differential windows of plasticity were identified for ERß and oxytocin receptor mRNA levels. These studies contribute significantly to our understanding of the molecular, neurobiological, and behavioral pathways through which variation in maternal behavior is transmitted from one generation to the next.

Flibanserin treatment increases appetitive sexual motivation in the female rat.

INTRODUCTION: Flibanserin is a mixed 5-HT1A agonist/5-HT2A antagonist that has been developed for the treatment of hypoactive sexual desire disorder in women. AIM: To assess the acute and chronic dose-response effects of flibanserin on measures of sexual desire and copulation in ovariectomized rats primed with estradiol benzoate (EB) alone or in combination with progesterone (P). METHODS: In Experiment 1, sexually experienced ovariectomized (OVX) rats at one testing site were rendered fully sexually receptive with EB + P priming and tested weekly with a sexually active male in bi-level pacing chambers following daily flibanserin treatment for 28 days. In Experiment 2, sexually experienced OVX rats at a different testing site received EB alone and were tested weekly with sexually active males following daily flibanserin treatment. MAIN OUTCOME MEASURES: Female appetitive behaviors (solicitations, hops and darts, anogenital investigations), defensive behaviors, pacing, lordosis, and male copulatory responses (intromissions and ejaculations) were measured during each 30-minute copulation test. RESULTS: Acute flibanserin or 1 week of chronic flibanserin treatment did not modify sexual responses in fully (EB + P) or partially (EB-alone) primed females. After 2 weeks of chronic treatment, fully primed females displayed significantly more solicitations than the three other groups. After 3 weeks of chronic treatment, a significant increase in female solicitations was observed in both hormone-treatment groups. CONCLUSION: This study shows the first evidence that chronic, but not acute, flibanserin treatment augments appetitive sexual behaviors in OVX female rats primed with EB + P or EB alone. Given the positive effect of flibanserin in clinical trials, these results confirm previous reports that solicitations in the female rat are a predictive animal model of human female sexual desire.

Tracing copper-thiomolybdate complexes in a prospective treatment for Wilson's disease.

Wilson's disease is a human genetic disorder which results in copper accumulation in liver and brain. Treatments such as copper chelation therapy or dietary supplementation with zinc can ameliorate the effects of the disease, but if left untreated, it results in hepatitis, neurological complications, and death. Tetrathiomolybdate (TTM) is a promising new treatment for Wilson's disease which has been demonstrated both in an animal model and in clinical trials. X-ray absorption spectroscopy suggests that TTM acts as a novel copper chelator, forming a complex with accumulated copper in liver. We have used X-ray absorption spectroscopy and X-ray fluorescence imaging to trace the molecular form and distribution of the complex in liver and kidney of an animal model of human Wilson's disease. Our work allows new insights into metabolism of the metal complex in the diseased state.

In vitro and in vivo noise analysis for optical neural recording.

Laser diodes (LD) are commonly used for optical neural recordings in chronically recorded animals and humans, primarily due to their brightness and small size. However, noise introduced by LDs may counteract the benefits of brightness when compared to low-noise light-emitting diodes (LEDs). To understand noise sources in optical recordings, we systematically compared instrument and physiological noise profiles in two recording paradigms. A better understanding of noise sources can help improve optical recordings and make them more practical with fewer averages. We stimulated lobster nerves and a rat cortex, then compared the root mean square (RMS) noise and signal-to-noise ratios (SNRs) of data obtained with LED, superluminescent diode (SLD), and LD illumination for different numbers of averages. The LED data exhibited significantly higher SNRs in fewer averages than LD data in all recordings. In the absence of tissue, LED noise increased linearly with intensity, while LD noise increased sharply in the transition to lasing and settled to noise levels significantly higher than the LED's, suggesting that speckle noise contributed to the LD's higher noise and lower SNRs. Our data recommend low coherence and portable light sources for in vivo chronic neural recording applications.

Proteasome activity deregulation in LEC rat hepatitis: following the insights of transcriptomic analysis.

LEC rats show spontaneous hepatitis and hepatocarcinoma development related to oxidative stress due to abnormal copper accumulation in the liver. We used DNA microarrays bearing 22,012 genes to investigate at the transcriptomic level the progression of the hepatitis in LEC rats in comparison to a control obtained from LEC rats treated with D-penicillamine, a copper chelating agent known to block hepatitis development. Multivariate statistical analyses as partial least square (PLS) regression between transcriptomic data and hepatitis markers in plasma led us to select 483 genes related to hepatitis development in these rats. After a complementary discriminant analysis (PLS-DA), 239 important genes for the separation between the different rat groups were selected. Gene ontology classification revealed an overrepresentation of genes involved in protein metabolism-related functions. More importantly, some genes implicated in proteasome pathway were upregulated. However, analysis of 20S proteasome activity showed that trypsin-like and peptidylglutamyl peptide hydrolase activities were diminished during hepatitis. Because oxidative stress is known to promote the inactivation of the proteasome complex, we propose the deregulation of the proteasome genes expression as a result of oxidative inactivation of proteasome activity during hepatitis in LEC rats. These results bring new insights in the hepatitis and the hepatocarcinogenesis development.

Down-regulation of intestinal multidrug resistance-associated protein 2 in long-evans cinnamon rats.

Wilson's disease is an inherited, autosomal recessive disorder of copper accumulation and toxicity. Lifelong chelation therapy is essential in all Wilson's disease patients. Intestinal absorption of some compounds is limited partly because they are preferentially transported in the secretory direction. Several ATP-binding cassette (ABC) transporters are expressed in the apical membrane of the small intestine and secrete various drugs into the lumen. In this study, we investigated the characteristics of the intestinal efflux ABC transporters in LEC rats. We found that the expression of multidrug resistance-associated protein 2 (Mrp2) in the jejunum of Long-Evans Cinnamon (LEC) rats, an animal model for Wilson's disease, is decreased.

A new amphiphilic derivative, N-{[4-(lactobionamido)methyl]benzylidene}-1,1-dimethyl-2-(octylsulfanyl)ethylamine N-oxide, has a protective effect against copper-induced fulminant hepatitis in Long-Evans Cinnamon rats at an extremely low concentration compared with its original form alpha-phenyl-N-(tert-butyl) nitrone.

An amphiphilic alpha-phenyl-N-(tert-butyl) nitrone (PBN) derivative, N-{[4-(lactobionamido)methyl]benzylidene}-1,1-dimethyl-2-(octylsulfanyl)ethylamine N-oxide (LPBNSH), newly synthesized from its original form PBN in hopes of clinical use, was intraperitoneally administered to Long-Evans Cinnamon (LEC) rats every 2 days at the concentrations of 0.1, 0.5, 1.0, and 2.0 mg/kg. We found that LPBNSH protected against copper-induced hepatitis with jaundice in LEC rats at concentrations of 0.1 and 0.5 mg/kg, which were extremely low compared with that of PBN. It also effectively prevented the loss of body weight, reduced the death rate, and suppressed the increase in serum aspartate aminotransferase and alanine aminotransferase values arising from fulminant hepatitis with jaundice at the same concentrations. Similar results were observed when PBN was administered at the concentration of 150 mg/kg. Immunohistochemical analysis of 8-hydroxy-2'-deoxyguanosine and measurement of thiobarbituric acid-reactive substances in the liver showed that LPBNSH largely suppressed the formation of these oxidative products at same concentrations. No difference in the abnormal accumulation of copper in the liver between the LPBNSH administered and control groups was observed. From these results, it was concluded that LPBNSH exhibited liver-protective effects against fulminant hepatitis with jaundice at ca. 1/1000, 500 the molar concentration of PBN and, therefore, was clinically promising.

Maturational arrest of thymocyte development is caused by a deletion in the receptor-like protein tyrosine phosphatase kappa gene in LEC rats.

The Long-Evans Cinnamon (LEC) rat has a spontaneous mutation, T helper immunodeficiency (thid), which causes a markedly reduced CD4(+) thymocyte population. Here we positionally clone the locus and identify a deletion in the gene encoding a receptor-like protein tyrosine phosphatase kappa (Ptprk) that led to complete loss of the transcript. The rat Ptprk gene exhibits 98% identity with the human and mouse counterparts and is expressed most abundantly in the CD4(-)CD8(-) double-negative stage. The downregulation of Ptprk in mouse immature thymocytes by RNA interference mimicked the thid phenotype. These results indicate that thid maps to the Ptprk locus and that functional Ptprk is crucial for lineage commitment or progression of CD4(+) T cells. We also found that Ptprk appears to function in parallel with or downstream of Th-POK/cKrox (also known as ZBTB7B), a master regulator of T cell lineage decision.

Inhibitory effects of fermented brown rice and rice bran on the development of acute hepatitis in Long-Evans Cinnamon rats.

To investigate the effects of fermented brown rice (FBRA) on the development of hereditary hepatitis in Long-Evans Cinnamon (LEC) rats, we compared the incidence and grades of acute hepatitis among rats fed 5% and 10% FBRA in the conventional diet and the conventional diet alone. Both the 5% and 10% FBRA-supplemented diets indicated a tendency to prevent the development of hepatitis, and the significant effect of 10% FBRA was observed until 16-17 weeks of age in the accumulated incidence and survival ratio compared with the unsupplemented conventional diet, although no significant difference was observed between 5% and 10% FBRA-supplemented diets. At the age of 12 weeks, which is just before the rats develop hepatitis, serum copper levels in rats fed either of the test diets were similar to those in rats fed the conventional diet. Furthermore, the copper concentration in liver tissue at 12 weeks of age was not changed by the test diet. These results suggest that FBRA has preventive effects on the development of hepatitis in LEC rats and may play an important role in protecting the liver against the free radicals induced by copper accumulation in the liver.

Development of liver regenerative therapy using glycoside-modified bone marrow cells.

Several recent studies have reported that bone marrow cells (BMCs) have the ability to generate functional hepatocytes. However, the efficiency at which BMC transplantation generates functional hepatocytes is rather low. We assumed that if BMCs accumulated directly in liver, the functional BMC-derived hepatocytes should increase efficiently. We tried to increase the accumulation of BMCs directly in liver through the interaction between hepatic asialoglycoprotein receptor and desialylated BMCs. Desialylated BMCs were produced with treatment of neuraminidase. Desialylated BMCs that expressed green fluorescent protein (GFP) were injected into Long Evans Cinnamon (LEC) rats, a human Wilson's disease model, intravenously. At 3 and 5 months after transplantation, GFP-expressing hepatocyte nodules appeared in the liver of these BMC-transplanted LEC rats. These findings suggest that the functional BMC-derived hepatocytes can be generated by the direct accumulation of BMCs and that this strategy is new BMC therapy for liver regeneration.

Zinc supplementation decreases hepatic copper accumulation in LEC rat: a model of Wilson's disease.

The effect of dietary zinc (Zn) supplementation on copper (Cu)-induced liver damage was investigated in Long-Evans Cinnamon rats (LEC), a model for Wilson's disease (WD). Four-week-old LEC (N=64) and control Long-Evans (LE) (N=32) female rats were divided into two groups; one group was fed with a Zn-supplemented diet (group I) and the other was given a normal rodent diet (group II). LEC rats were killed at 6, 8, 10, 12, 18, and 20 wk of age; the LE control rats were killed at 6, 12, 18, and 20 wk of age. Cu concentration in the liver was reduced in LEC rats fed the Zn-supplemented diet compared with LEC rats on the normal diet between 6 and 18 wk of age. Metallothionein (MT) concentration in the livers of LEC rats in group I increased between 12 and 20 wk of age, whereas hepatic MT concentration in LEC rats from group II decreased after 12 wk. Hepatocyte apoptosis, as determined by TUNEL, was reduced in Zn-supplemented LEC rats at all ages. Cholangiocellular carcinoma was observed only in LEC rats in group II at wk 20. These results suggest that Zn supplementation can reduce hepatic Cu concentration and delay the onset of clinical and pathological changes of Cu toxicity in LEC rats. Although the actual mechanism of protection is unknown, it could be explained by sequestration of dietary Cu by intestinal MT, induced by high dietary Zn content.

Efficacy of zinc supplementation in preventing acute hepatitis in Long-Evans Cinnamon rats.

OBJECTIVES: Long-Evans Cinnamon (LEC) rats are characterized by an abnormal hepatic deposition of copper (Cu) due to a lack of the Cu-transporter P-type adenosine triphosphatase: accordingly, the strain is a good animal model of Wilson's disease. The effect of oral zinc (Zn) acetate treatment on the development of acute hepatitis and the biochemical parameters of Cu-induced liver damage was studied in 5-week-old LEC rats (n=52). METHODS: Rats receiving 50 or 80 mg/ml/day Zn acetate by gavage and control rats receiving a daily dose of glucose solution 0.02 g/ml by gastric intubation were killed at 1, 2 or 8 weeks after the start of treatment. RESULTS: Treatment with Zn acetate resulted in the prevention of acute hepatitis: 10 of the 13 untreated rats developed signs and symptoms compatible with acute hepatitis between the 6th and 7th week of treatment. Tissue metallothionein (MT) significantly increased in the treated rats and positively correlated with Zn concentrations within the liver. Control rats had a significantly higher iron concentration in the liver and kidneys compared with supplemented rats, after both short- and long-term experiments. 8-hydroxy-2'-deoxyguanosine amounts were significantly lower in untreated rats. CONCLUSIONS: Zn acetate prevents acute hepatitis, by increasing tissue MT concentrations, reducing Cu absorption and interfering with Fe metabolism.

L-carnitine inhibits hepatocarcinogenesis via protection of mitochondria.

Hepatocellular carcinoma is usually preceded by chronic inflammation. However, the molecular mechanism in hepatocarcinogenesis is not well known. Recently, we reported that mitochondrial dysfunction plays an important role in hepatocarcinogenesis via the production of free radicals. Furthermore, we proved that L-carnitine effectively protects mitochondrial function in vivo. Therefore, we investigated whether long-term administration of L-carnitine could prevent hepatitis and subsequent hepatocellular carcinoma in Long-Evans Cinnamon rats that are often analyzed as a model of hepatocarcinogenesis. The results indicated that oxidative stress elicited from abnormally accumulated copper increased the amount of free fatty acids, thereby inducing mitochondrial dysfunction, resulting in cell death and enhanced secondary generation of reactive oxygen species, which were significantly inhibited by carnitine treatment. Finally, the occurrence of placental glutathione S-transferase-positive foci as a marker for preneoplastic lesions and hepatocarcinogenesis were significantly inhibited by L-carnitine. These facts suggest that mitochondrial injury plays an essential role in the development of hepatocarcinogenesis and that the clinical use of carnitine has excellent therapeutic potential in individuals with chronic hepatitis.

Relationship between metallothionine and zinc in the protection against DNA damage in zinc-treated Long-Evans Cinnamon rat liver.

The aims of the work presented here were to determine the effect of long term treatment with zinc (Zn) on both total metallothionine (MT) and, in particular, oxidized MT (MTox) concentrations in Long-Evans Cinnamon (LEC) rat liver. We also evaluated semi-quantitatively the cell death index using TUNEL assay as it is a useful method to localize the nuclear fragmentation occurring in oxidative stress conditions. The results demonstrate there were no statistically different MT concentrations between Zn-treated and untreated rats, whereas the Zn treatment was very effective in reducing the percentage of oxidized MT (MTox). MTox is not able to bind metals, so it does not perform its "scavenger" action against copper (Cu) accumulation in LEC rats. The intensity and quantity of fluorescent staining observed in untreated rat sections decreased compared to the treated ones. These findings suggest that in LEC rats one of zinc's roles is to protect from oxidative stress, however, its mode of action remains partially unknown: a hypothesis is competition for Cu binding sites. A new insight is that Zn induced MT can protect efficiently against DNA damage by free radicals.

High sensitivity of fibroblast cell lines derived from LEC rats to heat treatment.

A fibroblast cell line derived from LEC rat was approximately twofold more sensitive to heat treatment at 45 degrees C than were that from WKAH rat in terms of heating time required to attain 50% loss of survival in a colony forming assay. The present study was carried out for understanding the mechanism underlying the higher sensitivity of LEC rat cells to heat treatment. Although apoptosis was not found in WKAH rat cells, the percentages of apoptotic cells in LEC rat cells significantly increased after heat treatment. LEC rat cells showed significantly lower sensitivity in induction of cell death and apoptosis to ceramide, a lipid signaling molecule that is associated with heat-induced apoptosis, than did WKAH rat cells. SP600125, an inhibitor of JNK suppressed the induction of cell death in both heated LEC and WKAH rat cells, but SB203580, an inhibitor of p38 mapk, did not. The relative surviving fractions of heated LEC and WKAH rat cells in the presence of both SB203580 and SP600125 were higher than those of cells in the presence of SP600125 alone. The amounts of hsp70 protein in WKAH rat cells increased from 4 to 12 hr after heat treatment, but did not in LEC rat cells. These results suggest that higher thermosensitivity in the fibroblast cell line from LEC rat is due to low inducibility of hsp70 protein after heat treatment.