mL) - Nature

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+TAA. Cx32 +/+ 2APB. +TAA. TAA (mg/mL). -. 1003 ± 28. 995 ± 36. TASO (mg/mL). -. 162 ± 18. 148 ± 21. Nature Biotechnology: doi:10.1038/nbt.2089 ...
Cx32 +/+ Saline

Cx32 +/+ TAA

Cx32 -/- TAA

TAA (mg/mL)

-

986 ± 31

983 ± 27

TASO (mg/mL)

-

187 ± 13

178 ± 9

Cx32 +/+ DMSO

Cx32 +/+ DMSO +TAA

Cx32 +/+ 2APB +TAA

TAA (mg/mL)

-

1003 ± 28

995 ± 36

TASO (mg/mL)

-

162 ± 18

148 ± 21

Nature Biotechnology: doi:10.1038/nbt.2089

Supplementary Figure 1. Deficiency in Cx32 does not affect xenobiotic metabolism. HPLC analysis was performed to demonstrate that the protection against TAA hepatotoxicity in Cx32-/mice and Cx32+/+ treated with 2APB was not a result of defective drug metabolism in the liver. A reverse-phase HPLC assay was used to quantify the serum concentrations of TAA and its toxic metabolite, TASO, in Cx32+/+ or Cx32-/- mice treated with saline, TAA (1000 mg/kg), DMSO vehicle control (DMSO, .1 mL/kg) plus TAA (1000 mg/kg), 2APB (20 mg/kg) plus TAA (1000 mg/kg), or 2APB (20 mg/kg) for 90 minutes. Standards were prepared by including known amounts of TAA and TASO in serum from untreated mice. Equal concentrations of TAA and TASO were found in sera of Cx32+/+ and Cx32-/- mice treated with TAA, as well as in sera of Cx32+/+ mice treated with 2APB.

Nature Biotechnology: doi:10.1038/nbt.2089

Nature Biotechnology: doi:10.1038/nbt.2089

Supplementary Figure 2. Phase I and II drug metabolism efficiency is similar in Cx32+/+ and Cx32-/- mice. Whole livers were excised from untreated Cx32+/+ and Cx32-/- mice and processed for analysis. (a) Q-PCR for cytochrome P450 enzymes Cyp1a1, Cyp1a2, Cyp2e1, Cyp2b10, and Cyp32 reveals approximately equal expression in Cx32+/+ and Cx32-/- livers. (b) Analysis of GST activity shows equal activity in Cx32+/+ and Cx32-/- livers. (c) Total GSH content was found to be similar in Cx32+/+ and Cx32-/- livers.

Nature Biotechnology: doi:10.1038/nbt.2089

Nature Biotechnology: doi:10.1038/nbt.2089

Supplementary Figure 3. Drug-induced hepatotoxicity is dependent on oxidative stress. Free radical scavenger DMSO1 and anti-oxidant NAC were utilized to demonstrate the dependency of TAA- and APAP-induced hepatotoxicity, respectively, on oxidative stress. (a, b) Serum ALT levels and H&E liver histology (original magnification 10X; scale bar = 400 µm) from wildtype mice 24 hours after treatment with DMSO (1 mL/kg) alone, saline (1 mL/kg) plus TAA (200 mg/kg), or DMSO (0.1 or 1 mL/kg) plus TAA (200 mg/kg) (*P