Potential roles of hepatic heat shock protein 25 and 70i in protection of mice against acetaminophen-induced liver injury

The aim of the present study was to assess the contribution of the level of expression of heat shock protein 25 (HSP25), 60 (HSP60), 70 (HSC70) and 70i (HSP70i) in mouse livers after a lethal dose of acetaminophen (APAP) to their survival. We examined changes in survival ratio, plasma APAP level and alanine aminotransferase (ALT) activity, and hepatic reduced glutathione (GSH), HSP25, HSP60, HSC70 and HSP70i levels following treatment of mice with APAP (500 mg/kg, p.o.). The plasma APAP level increased rapidly, and reached a maximum 0.5 h after APAP treatment. Hepatic GSH decreased rapidly, and was almost completely depleted 1 h after APAP treatment. Plasma ALT activity, an index of liver injury, significantly increased from 3 h onwards after APAP treatment. The survival ratios 9 h, 24 h and 48 h after APAP treatment were 96%, 38% and 36%, respectively. We found a remarkable difference in the patterns of hepatic HSP25 and HSP70i induction in mice that survived after APAP treatment. HSP70i levels increased from 1 h onwards after APAP treatment in a time-dependent manner, and reached a maximum at 9 h. In contrast, HSP25 could be detected just 24 h after APAP treatment, and maximal accumulation was observed at 48 h. Other HSPs examined were unchanged. Notably, the survival ratio dropped by only 2% after HSP25 expression. Recently, a novel role for HSP25 as an anti-inflammatory factor was suggested. We have already shown that 48-h treatment with APAP induces severe centrilobular necrosis with inflammatory cell infiltration in mouse livers. Taken together, the level of expression of hepatic HSP25 may be a crucial determinant of the fate of mice exposed to APAP insult.     

Functional analysis of the SGNP I in the pupal diapause of the oriental tobacco budworm, Helicoverpa assulta (Lepidoptera: Noctuidae)

Helicoverpa assulta suboesophageal ganglion neuropeptide I (Has-SGNP I) is a 24-amino acids peptide amide, which shows 62.5% similarity with the diapause hormone of Bombyx mori (Bom-DH). It has been demonstrated that embryonic diapause is induced by DH in B. mori. Injection of synthetic amidated Has-SGNP I terminated pupal diapause in a dose-dependent manner. Therefore, Has-SGNP I might be referred to a "diapause termination hormone" in H. assulta (Has-DTH). The maximal dose of Has-DTH for diapause termination was 1.0 microg and the half-maximal dose 0.4 microg. The time required for diapause termination of Has-DTH was 2-3 days longer than that of 20-hydroxyecdysone. During the pupal stage, DTH mRNA content in the SGs of nondiapausing pupae was always higher than in diapausing pupae using the combined method of quantitative RT-PCR and Southern blot. DTH gene also expressed at a low level while diapausing pupae were chilled at 4 degrees C, but increased rapidly and largely after being transferred to 25 degrees C. Using a competitive ELISA, Has-DTH-like immunoreactivity in the haemolymph showed the same pattern as that of Has-DTH gene expression. Those results indicated that Has-DTH gene expression was related to diapause development and could be activated by low temperature. Has-DTH might be useful to elucidate the mechanism of diapause termination in pupal diapause species.     

Hepatic Mrp4 induction following acetaminophen exposure is dependent on Kupffer cell function

During acetaminophen (APAP) hepatotoxicity, increased expression of multidrug resistance-associated proteins 2, 3, and 4 (Mrp2-4) occurs. Mrp4 is the most significantly upregulated transporter in mouse liver following APAP treatment. Although the expression profiles of liver transporters following APAP hepatotoxicity are well characterized, the regulatory mechanisms contributing to these changes remain unknown. We hypothesized that Kupffer cell-derived mediators participate in the regulation of hepatic transporters during APAP toxicity. To investigate this, C57BL/6J mice were pretreated with clodronate liposomes (0.1 ml iv) to deplete Kupffer cells and then challenged with APAP (500 mg/kg ip). Liver injury was assessed by plasma alanine aminotransferase and hepatic transporter protein expression was determined by Western blot and immunohistochemistry. Depletion of Kupffer cells by liposomal clodronate increased susceptibility to APAP hepatotoxicity. Although increased expression of several efflux transporters was observed after APAP exposure, only Mrp4 was found to be differentially regulated following Kupffer cell depletion. At 48 and 72 h after APAP dosing, Mrp4 levels were increased by 10- and 33-fold, respectively, in mice receiving empty liposomes. Immunohistochemistry revealed Mrp4 staining confined to centrilobular hepatocytes. Remarkably, Kupffer cell depletion completely prevented Mrp4 induction by APAP. Elevated plasma levels of TNF-alpha and IL-1beta were also prevented by Kupffer cell depletion. These findings show that Kupffer cells protect the liver from APAP toxicity and that Kupffer cell mediators released in response to APAP are likely responsible for the induction of Mrp4.     

Differential gene expression during early embryonic development in diapause and non-diapause eggs of multivoltine silkworm Bombyx mori

Quantification of the differential expression of metabolic enzyme and heat-shock protein genes (Hsp) during early embryogenesis in diapause and non-diapause eggs of the silkworm B. mori was carried out by semi-quantitative RT-PCR. Data analysis revealed that, the phosphofructokinase (PFK) expression started at a higher level in the early stage (6 h after oviposition) in non-diapause eggs, while in diapause induced eggs, it started at a lower level. However, the PFK gene expression in diapause eggs was comparatively higher than in non-diapause eggs. PFK facilitates use of carbohydrate reserves. The lower level of PFK gene expression in the early stage of diapause induced eggs but comparatively higher level of expression than in non-diapause eggs is due to enzyme inactivation via protein phosphorylation during early embryogenesis followed by de-phosphorylation in later stage. The sorbitol dehydrogenase-2 (SDH-2) gene was down regulated in diapause induced eggs up to 24 h and its expression levels in diapause induced eggs coincided with that of PFK gene at 48h in non-diapause eggs. During carbohydrate metabolism, there is an initial temporary accumulation of sorbitol which acts as protectant. The down regulation of SDH-2 gene during the first 24 hours in diapause induced eggs was due to the requirement of sorbitol as protectant. However, since the diapause process culminates by 48 h, the SDH-2 gene expression increased and coincided with that of PFK gene expression. The trehalase (Tre) gene expression was at a lower level in diapause induced eggs compared to non-diapausing eggs. The induction of Tre activity is to regulate uptake and use of sugar by the tissues. The non-diapause eggs revealed maximum expression of GPase gene with major fluctuations as well as an overall higher expression compared to diapause induced eggs. The diapause process requires less energy source which reflects lower activity of the gene. Heat shock protein (Hsp) genes (Hsp20.4, 40, 70, and 90) revealed differential levels of expression in both the eggs at all stages of embryonic development. The present study thus provides an overview of the differential expression levels of metabolic enzyme and Hsp genes in non-diapause and diapause induced eggs of multivoltine silkworm B. mori within 48 h after oviposition, confirming the major role of in early embryogenesis.     

Vascular endothelial growth factor and hepatocyte regeneration in acetaminophen toxicity

VEGF or VEGF-A is a major regulator of angiogenesis and has been recently shown to be important in organ repair. The potential role of VEGF in acetaminophen (APAP)-induced hepatotoxicity and recovery was investigated in B6C3F1 male mice. Mice were treated with APAP (300 mg/kg ip) and killed at various time points that reflect both the acute and recovery stages of toxicity. VEGF-A protein levels were increased 7-fold at 8 h and followed the development of hepatotoxicity. VEGF receptor 1, 2, and 3 (VEGFR1, VEGFR2, and VEGFR3, respectively) expression increased throughout the time course, with maximal expression at 48, 8, and 72 h, respectively. Treatment with the VEGF receptor inhibitor SU5416 (25 mg/kg ip at 3 h) had no effect on toxicity at 6 or 24 h. In further studies, the role of SU5416 on the late stages of toxicity was examined. Treatment of mice with APAP and SU5416 (25 mg/kg ip at 3 h) resulted in decreased expression of PCNA, a marker of cellular proliferation. Expression of platelet endothelial cell adhesion molecule, a measure of small vessel density, and endothelial nitric oxide synthase (NOS), a downstream target of VEGFR2, were increased at 48 and 72 h following toxic doses of APAP, and treatment with SU5416 decreased their expression. These data indicate that endogenous VEGF is critically important to the process of hepatocyte regeneration in APAP-induced hepatotoxicity in the mouse.     

Stem cell factor and c-kit are involved in hepatic recovery after acetaminophen-induced liver injury in mice

Stem cell factor (SCF) and its receptor c-kit are important in hematopoiesis and cellular proliferation. c-kit has also been identified as a cell surface marker for progenitor cells. We have previously shown that there is a large reservoir of hepatic SCF, and this molecule plays a significant role in liver regeneration after 70% hepatectomy. In the current study, we further examined the expression of SCF and c-kit in acetaminophen (APAP)-induced liver injury in C57BL/6J mice or SCF-deficient sl-sld mice and their appropriate wild-type controls. Following APAP-induced liver injury, c-kit mRNA expression increased, with peak levels detected 48 h postinjury. Hepatic SCF mRNA levels after APAP injury were also increased, with peak levels seen 16 h post-APAP. The mortality rate in SCF-deficient mice treated with APAP was significantly higher than that of wild-type mice; furthermore, administration of exogenous SCF significantly reduced the mortality of APAP-treated wild-type mice. Bromodeoxyuridine incorporation experiments showed that SCF significantly increased hepatocyte proliferation at 48 and 72 h in APAP-treated mice. SCF inhibited APAP-induced hepatocyte apoptosis and increased Bcl-2 and Bcl-xL expression, suggesting that this decrease in hepatocyte apoptosis is mediated through Bcl-2 and Bcl-xL. In summary, SCF and c-kit expression was increased after APAP-induced liver injury. Administration of exogenous SCF reduces mortality in APAP-treated mice, increases hepatocyte proliferation, and prevents hepatocyte apoptosis induced by APAP, suggesting that these molecules are important in the liver's recovery from these injuries.     

Interleukin 6 and hepatocyte regeneration in acetaminophen toxicity in the mouse

To determine the importance of IL-6 in acetaminophen (APAP) toxicity, wild type (WT) and IL-6 knock out (KO) mice were dosed with APAP (300 mg/kg i.p.) and sacrificed at 4 and 24h. No differences were found between the two groups by analysis of serum AST levels or histopathology. Also, the relative amounts of APAP protein binding and nitrotyrosine formation were equal. Subsequently, WT and KO mice were dosed with APAP (300 mg/kg i.p.) and sacrificed at 24, 48, and 72 h. AST normalized by 48 h in the WT mice, but not until 72 h in the KO mice. The severity of the histopathological alterations was comparable in the two groups of mice; however, fewer regenerating hepatocytes were present in the KO mice. Immunohistochemistry for proliferating cell nuclear antigen (PCNA) showed reduced staining in the KO mice. Pretreatment of KO mice with IL-6 lowered AST and normalized PCNA staining in the IL-6 KO mice. These data suggest that IL-6 is important in hepatocyte regeneration following APAP toxicity in the mouse.     

Photoperiodic induction of pupal diapause in the flesh fly,Sarcophaga crassipalpis: embryonic sensitivity

Summary The last two days of embryonic development are crucial in programming pupal diapause in the flesh fly,Sarcophaga crassipalpis. Short daylength (greater than 10 1/2h of darkness) during this interval permits expression of diapause while long daylength during this brief sensitive stage eliminates the potential for diapause. Length of scotophase rather than photophase programs the diapause although three hours of light is needed to separate tandem dark periods. Early in the scotophase, photosensitivity is restricted to blue light (less than 540 nm). The scotophase can be divided into 4 phases according to the effect of light breaks on diapause expression. During Phase I (0–6 h after scotophase onset) embryos are highly sensitive to light interruption and diapause is effectively eliminated. A period of insensitivity to light, Phase II, extends from 6–hh after onset of scotophase. Light breaks at 10–11h coincide with the critical scotophase length and result in a partial reduction of diapause. In Phase IV, the scotophase reaction is complete and diapause competence is preserved even in the presence of light. Although light breaks result in elimination of diapause throughout Phase I, recovery time from a 1 h light break (length of darkness needed to counter the effect of a light break) differs dramatically depending upon when the light break is presented. Early in Phase I (0–3h) recovery from light interruption is rapid, while late in Phase I (4–6h), the effects of light are not readily reversible. The scotophase reaction thus appears to follow a step-wise progression rather than represent a simple linear response. We present a molecular model that could account for the dynamics of the scotophase reaction.     

即时浸酸显著降低滞育家蚕卵的还原型与氧化型谷胱甘肽比值

即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 显著提高了家蚕卵H₂O₂含量。还原型谷胱甘肽（reduced glutathione, GSH）与氧化型谷胱甘肽（oxidized glutathione, GSSG）的比值是一种氧化胁迫状态的动态指标。为了调查即时浸酸是否造成滞育家蚕卵氧化胁迫, 本研究利用分光光度法分别测定了滞育家蚕卵和5 min即时浸酸滞育家蚕卵中GSH和GSSG含量以及谷胱甘肽转移酶（glutathione-S-transferase, GST）活性。结果表明: 处理后24 h, 即时浸酸处理家蚕卵的总谷胱甘肽（GSH+2GSSG）含量、 GSH含量、 GSSG含量、 GSH/GSSG比值和GST活性分别相当于同期滞育家蚕卵的204%, 78%, 550%, 14%和97%。据此推测, 即时浸酸在阻止滞育发动的同时, 可能通过促进GSH氧化为GSSG, 而显著降低了GSH/GSSG比值, 使家蚕卵处于过氧化状态。     

IFN-gamma-inducible protein-10 (CXCL10) is hepatoprotective during acute liver injury through the induction of CXCR2 on hepatocytes.

IFN-gamma-inducible protein-10 (IP-10/CXCL10) is a non-ELR-CXC chemokine that is present during various forms of acute and chronic liver injury. The purpose of this study was to explore the role of IP-10 during acute liver injury induced by acetaminophen (APAP). After a 400 mg/kg APAP challenge in fasted CD-1 mice, immunoreactive levels of IP-10 were dramatically elevated in the serum within 8 h. CXCR3, the receptor for IP-10, was up-regulated in the liver. Mice that received an i.v. injection of rIP-10 10 h after APAP challenge exhibited a dramatic reduction in alanine aminotransferase 8 h later. Histologic analysis confirmed that the delayed IP-10 therapy dramatically improved the appearance of the liver when examined 48 h after APAP. The therapeutic effect of IP-10 was associated with a marked increase in CXCR2 expression on hepatocytes. Neutralization of CXCR2 during IP-10 therapy resulted in an abrogation of the hepatoprotective effect of IP-10. Furthermore, IP-10 treatment of cultured hepatocytes stimulated a CXCR2-dependent proliferative response. In conclusion, IP-10 has a hepatoregenerative effect in a murine model of acute liver injury that is dependent on its up-regulation of CXCR2 on hepatocytes.     

Effects of acetaminophen on hepatic gene expression in mice

Acetaminophen (APAP) is one of the most commonly used drugs for the safe and effective treatment of fever and pain. However, it is a well-established hepatotoxin. The objective of this study was to identify alternation in various genes in liver of mice after administration of low and high doses of APAP. Male C57BL/6J mice received APAP (30 or 300 mg/kg, i.p.). They were sacrificed after 6 hr and 24 hr for assessment of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total RNA isolation, cDNA microarray analysis and histopathological analysis of liver injury. Low dose of APAP did not cause hepatotoxicity in mice. However, it was toxic at a high dose. Using microarray technology, we selected changed genes more than 1.5 fold. Gene expression changes were recorded even at a low dose treatment with APAP. Six (6) hr after APAP treatment at low dose, 6 genes were up-regulated and 25 genes were down-regulated. However, 24 hr after treatment at low dose 8 genes were up-regulated and 34 genes were down-regulated. 6 hr after of high dose treatment 29 genes were down-regulated and none was up-regulated. A 24 hr treatment with high dose up-regulated 6 genes and down-regulated 18 genes. These expression patterns provide information on high versus low dose mechanisms of APAP toxicity. Gene expression signatures recorded after a nontoxic dose of APAP strongly support the validity of gene expression changes as meaningful markers of hepatotoxicity.     

Correlation of oxygen consumption, cytochrome c oxidase, and cytochrome c oxidase subunit I gene expression in the termination of larval diapause in the bamboo borer, Omphisa fuscidentalis

The moth Omphisa fuscidentalis (Lepidoptera, Pyralidae) is a univoltine insect with a larval diapause period lasting up to 9 months. We studied changes in O(2) consumption in conjunction with cytochrome c oxidase activity and cytochrome c oxidase subunit I (cox1) gene expression. O(2) consumption changed within a day, showing a supradian rhythm with a ca.12-h cycle at 25 degrees C. During the first two-thirds of the diapause period, from October to March, O(2) consumption was constant until January and then increased by March. Topical application of methoprene, a juvenile hormone analog (JHA), to diapausing larvae terminated the diapause and was associated with an increase in O(2) consumption rate at diapause termination. In JHA-treated larvae, cytochrome c oxidase activity in fat bodies was high at the beginning of the prepupal period and highest at pupation. cox1 expression in fat bodies displayed a transient peak 8 days after JHA application and peaked in the prepupal period. Taken together, our results show that the break of diapause by JHA is associated with the activation of cox1, bringing about an increase in cytochrome c oxidase activity, followed by an increase in O(2) consumption rate.     

Effects of photoperiod,temperature, food and relative humidity on the induction of diapause in the predatory miteAmblyseius potentillae

Aspects of the induction of diapause were studied in a Dutch strain of the phytoseiid miteAmblyseius potentillae. The photoperiodic response curve was of the long-day type, with a sharply defined critical daylength of 14.5 h. Critical daylength varied only little at temperatures between 15.0 and 22.5°C.All post-embryonic and possibly even late-embryonic stages of development were found to be sensitive to photoperiod; sensitivity appeared to be maximal during the protonymphal stage.It is shown that -carotene is necessary for some early step in the physiological mechanism of photoperiodic induction, and not (or not exclusively) for the expression of the diapause response.Two points of sensitivity to light could be demonstrated in the nights ofld 1311 andld 1212 long-night regimes, using 1-h night interruptions. These results are similar to those obtained in lightbreak experiments with spider mites and insects. However, no effect was found with light interruptions applied during the dark phase of anld 1014 long-night regime.In resonance experiments with a constant photophase (12 h) and a variable scotophase, a weak rhythmic response was found at 22.5°C; at 19.0°C this effect was completely absent.The relative humidity experienced by the mites during diapause induction as well as during diapause development influenced the rate of diapause completion under long days (ld 168). Diapause duration appeared to be shortest when the mites experienced low relative humidity (35±5%) during diapause induction and high relative humidity (75±5%) during diapause termination, and longest under the reverse conditions.     

Influence of acetaminophen and trichloroethylene on liver cytochrome P450-dependent monooxygenase system

The aim of the study was to evaluate the effect of acetaminophen (APAP) and/or trichloroethylene (TRI) on the liver cytochrome P450-dependent monooxygenase system, CYP2E1 and CYP1A2 (two important P450 isoforms), and liver glutathione (GSH) content in rats. Rats were given three different doses of APAP (250, 500 and 1000 mg/kg b...) and then the above-mentioned parameters were measured for 48 h. The lowest APAP dose produced small changes in the cytochrome P450 content of liver. At 500 mg/kg APAP increased the cytochrome P450 content to 230% of the control. The inductive effect was seen at 1000 mg/kg dose but at 24 h and later. NADPH-cytochrome P450 reductase activity was the highest after the lowest dose of APAP, while after the highest dose it was equal to the control value. TRI increased both the cytochrome P450 content and the NADPH-cytochrome P450 reductase activity. When TRI was combined with APAP, both these parameters increased in the first hours of observation, but they returned to the control values at 24 h. When APAP was given at 250 mg/kg, GSH levels decreased to 55% of the control at 8 h and returned to the control values at 24 h. The higher doses of APAP decreased GSH levels more than the lowest dose, but after 24 h GSH levels did not differ from those of the control. When TRI was given at 250 mg/kg, the GSH levels decreased to 68% of the control at 2 h and then they increased gradually and tended to exceed the control values at 48 h. The effect of TRI combined with APAP on the level of GSH was virtually the same as that of APAP alone given at 500 mg/kg.     

Juvenile hormone changes associated with diapause induction, maintenance, and termination in the beet webworm, Loxostege sticticalis (Lepidoptera: Pyralidae)

At 22°C and under a long-day photoperiod of L:D 16:8, all the last fifth instar Loxostege sticticalis larvae undergo prepupal stage and pupate without diapause. Under a short-day photoperiod of L:D 12:12, in contrast, they all enter diapause with approximately 36 days diapause maintenance and then terminate diapause spontaneously, although only 44% of the larvae terminated diapause successfully. Changes in hemolymph juvenile hormone (JH I) titers of diapause-destined larvae across diapause induction, maintenance and termination were examined using HPLC, and were compared with those of non-diapause-destined larvae from the fifth instar through pupation. JH I titer of the earliest fifth instar diapause-destined larvae remained at a high level with a peak of 220.4 ng/ml, though it decreased continuously to a minimum of 69.0 ng/ml on day 5 in the fifth instar when the larvae stopped feeding to enter diapause. During the diapause maintenance, JH I titer of the mature larvae increased significantly and maintained a high level until day 31 in prepupae. JH I titer declined and fluctuated at low level from 5 days before pupation. In contrast, JH I titer of both the fifth instar non-diapause-destined larvae and prepupae remained and fluctuated at low level consistently, as well as decreased before pupation. These results indicate that diapause induction and maintenance in this species might be a consequence of high JH, whereas diapause termination can be attributed to low JH titer, which was in agreement with the hormonal regulation observed in many other larval-diapausing insects.