Detection of anti-isoniazid and anti-cytochrome P450 antibodies in patients with isoniazid-induced liver failure

Abstract

Isoniazid (INH)-induced hepatotoxicity remains one of the most common causes of drug-induced idiosyncratic liver injury and liver failure. This form of liver injury is not believed to be immune-mediated because it is not usually associated with fever or rash, does not recur more rapidly on rechallenge, and previous studies have failed to identify anti-INH antibodies (Abs). In this study, we found Abs present in sera of 15 of 19 cases of INH-induced liver failure. Anti-INH Abs were present in 8 sera; 11 had anti-cytochrome P450 (CYP)2E1 Abs, 14 had Abs against CYP2E1 modified by INH, 14 had anti-CYP3A4 antibodies, and 10 had anti-CYP2C9 Abs. INH was found to form covalent adducts with CYP2E1, CYP3A4, and CYP2C9. None of these Abs were detected in sera from INH-treated controls without significant liver injury. The presence of a range of antidrug and autoAbs has been observed in other drug-induced liver injury that is presumed to be immune mediated.

Conclusion: These data provide strong evidence that INH induces an immune response that causes INH-induced liver injury.

Conflict of interest statement

Conflict of Interest:

No conflict of interest.

© 2014 by the American Association for the Study of Liver Diseases.

Figures

Figure 1

Western blot of lysozyme (L) or lysozyme modified by INA-NHS (L-INH), which mimics the covalent binding of the reactive metabolite of INH. Detection was with an anti-INH antibody as previously described (7).

Figure 2

Detection of anti-INH antibodies by western blotting. L = lysozyme and L-INH = INH-modified lysozyme. Serum was diluted 1:400; #1-0 = control serum from a patient at baseline, #1–2 = serum from the same patient 98 days after initiation of therapy (ALT = 144 U/L). ALF-19 and ALF-3 are sera from two patients with INH-induced liver failure.

Figure 3

Detection of anti-INH antibodies by ELISA and western blotting. A) Baseline sera before initiation of INH therapy (Baseline), sera from patients who were treated with INH but did not develop significant hepatotoxicity (Prophylaxis), sera from patients who developed INH-induced liver failure but did not test positive for anti-INH antibodies (Liver Failure −) and sera from patients who had liver failure and tested positive for anti-INH antibodies (Liver Failure +) were diluted 1:1000. The ELISA plate was coated either with lysozyme (L) or INH-modified lysozyme (L-INH); the ratio of OD absorbance from L-INH/L was used to determine the presence of anti-INH antibodies. B) To determine if the antibodies in the sera from the 8 patients that tested positive for anti-INH antibodies were, in fact, specific for INH, the sera were preincubated with 200 μM INH for 1 hour at 4 °C (+INH) and this blocked the binding. C) The presence of anti-INH antibodies in the remaining 6 serum samples that tested positive for anti-INH antibodies by ELISA were confirmed by western blotting. Either lysozyme (L) or INH-modified lysozyme (L-INH) was loaded on a gel and transferred into a nitrocellulose membrane as described in the Methods section. Poncau S staining is shown as the loading control. Serum was diluted 1:400; + INH = serum was preincubated with 1 mM INH at 4 °C for 1 hour. Values represent Mean ± S.E. Statistically significant from control **p

Figure 4

Detection of anti-CYP antibodies in the serum from patients treated with INH. Control sera were from patients before starting INH (Baseline), sera from patients treated with INH but without significant liver injury (Prophylaxis), sera from patients with INH-induced liver failure but without anti-CYP antibodies (Liver Failure (−), sera from patients with INH-induced liver failure that do have anti-CYP antibodies (Liver Failure (+), or sera from all of the patients who had INH-induced liver failure (All Liver Failure). A) autoantibodies against CYP2E1. B) antibodies against INH-modified CYP2E1, C) autoantibodies against CYP3A4. D) autoantibodies against CYP2C9. Values represent Mean ± S.E. Statistically significant from control **p

Figure 5

In vitro covalent binding of INH to CYP2E1, CYP2C9, or Cyp3A4. Each CYP isozyme was incubated with INH (100 μM) in the presence or absence of an NADPH regenerating system. Rabbit, anti-INH antibody was used to detect covalent of INH to CYP isozyme as previously described (7).