CREBH Determines the Severity of Sulpyrine-Induced Fatal Shock

<div><p>Although the pyrazolone derivative sulpyrine is widely used as an antipyretic analgesic drug, side effects, including fatal shock, have been reported. However, the molecular mechanism underlying such a severe side effect is largely unclear. Here, we report that the transcription factor CREBH that is highly expressed in the liver plays an important role in fatal shock induced by sulpyrine in mice. CREBH-deficient mice were resistant to experimental fatal sulpyrine shock. We found that sulpyrine-induced expression of cytochrome P450 2B (CYP2B) family genes, which are involved in sulpyrine metabolism, in the liver was severely impaired in CREBH-deficient mice. Moreover, introduction of CYP2B in CREBH-deficient liver restored susceptibility to sulpyrine. Furthermore, ectopic expression of CREBH up-regulated CYP2B10 promoter activity, and <em>in vivo</em> knockdown of CREBH in wild-type mice conferred a significant resistance to fatal sulpyrine shock. These data demonstrate that CREBH is a positive regulator of CYP2B in response to sulpyrine administration, which possibly results in fatal shock.</p> </div

RNAi-mediated CREBH-knockdown mice are resistant to the sulpyrine shock.

<p>(A) Livers of wild-type mice transfected with RNAi vectors for CREBH (n = 4) or negative control vectors (n = 4) were taken at 24 hr after transfection. Gene expression of CREBH was analysed by a quantitative RT-PCR assay. ***, P<0.001. (B, C) Wild-type mice transfected with RNAi vectors for CREBH (n = 14) or negative control vectors (n = 14) were intraperitoneally injected with 2.7 mg/g of sulpyrine at 24 hr after transfection. Survival rate was monitored for 48 hr. *, P<0.05. Sera were taken at 2 hr 40 min after sulpyrine injection. Serum concentrations of 4-AA and 4-FAA were measured by a HPLC assay. ***, P<0.001. Data are representative of two (A, C) independent experiments or pooled from three (B) independent experiments.</p

Cleavage of CREBH and its translocation into nucleus were induced by sulpyrine.

<p>(A) 293T cells were transiently transfected with 2 µg HA-tagged hCREBH-F expression vectors, and they were cultured with sulpyrine (2 mM) or tunicamycin (5 µg/ml) for 6 hr. Whole-cell lysates were prepared and the size of CREBH was analysed by western blot analysis using anti-CREBH monoclonal (upper panel) and anti-actin antibodies (lower panel). **, CREBH-full ; *, CREBH-ΔC. (B) 293T cells were transiently transfected with 2 µg HA-tagged hCREBH-F expression vectors, and they were cultured with sulpyrine (2 mM) or tunicamycin (5 µg/ml) for 6 hr. The cell lysates of nuclear fractions were prepared. The nuclear translocation of CREBH was analyzed by western blot analysis using anti-CREBH monoclonal (upper panel) and anti-Sp1 monoclonal antibodies (lower panel). (C) 293T cells were transiently transfected with HA-tagged hCREBH-F expression vectors and HA-tagged hCREBH-ΔC expression vectors, and they were cultured with mock, sulpyrine (2 mM) and tunicamycin (5 µg/ml) for 4 hr. Cells were stained with anti-HA mouse antibody, then stained with Alexa Fluor 594-conjugated anti-mouse IgG (red) together with DAPI (blue). Stained cells were analysed using a confocal microscope. Bars, 10 µm. Data are representative of three (A) and two (B,C) independent experiments.</p

Sulpyrine treatment causes ER stress.

<p>(A) Huh7 cells were transiently transfected with luciferase reporter plasmids containing the CYP2B10 promoter together with control, hCREBH-F, or hCREBH-ΔC expression vector. Relative luciferase activities were shown as fold increases over the background levels shown by lysates prepared from control vector-transfected cells. Error bars are means ± S.D. of triplicates. (B) Huh7 cells were transiently transfected with luciferase reporter plasmids containing the indicated length CYP2B10 promoter together with control or hCREBH-F expression vector. Relative luciferase activities were shown as fold increases over the background levels shown by lysates prepared from control vector-transfected cells. Error bars are means ± S.D. of triplicates. (C) Wild-type mice were intraperitoneally injected with 2.7 mg/g of sulpyrine (n = 3) or 200 µg/kg tunicamycin (n = 3) or vehicle alone (control) (n = 3). At 2 hr 40 min after injection, hepatocytes were taken from these mice by a perfusion apparatus. Gene expression of CHOP, spliced XBP1, and CYP2B10 was measured by a quantitative RT-PCR assay. **, P<0.01; ***, P<0.001. Data are representative of three (A, B) and two (C) independent experiments.</p

The blood concentration of 4-AA is involved in sulpyrine-induced shock.

<p>(A) Calibration curves for relationship between the administration volumes of 4-AA, 4-FAA and the concentrations of in the sera. (B) Wild-type mice (n = 5) were intraperitoneally injected with indicated volumes of 4-AA and 4-FAA. Survival rate was monitored for 180 min. (C) Wild-type mice (n = 5) were intraperitoneally injected with indicated volumes (0, 0.5, 0.9, 1.0, 1.2, 1.3, 1.4, and 1.5 mg/g) of 4-AA and 4-FAA. Survival rate was monitored for 180 min. Data are representative of two (A–C) independent experiments.</p