In Reply:-Drs. Njoku, Pohl, and Martin state that our article is “very misleading, contains statements inconsistent with the results and those of previously published articles, and contains discrepancies that are misleading.” We disagree and believe they misrepresent the paper and its predecessor. Drs. Njoku, Pohl, and Martin apparently do not disagree with the fundamental conclusion of the investigation, that compound A effects appear similar whether it is administered by inhalation or intraperitoneal injection. Rather, they criticize wording in the abstract, which was restricted by Anesthesiology to 250 words.
For example, we summarized our previous results, that probenecid and aminooxyacetic acid (AOAA) diminished compound A nephrotoxicity. Drs. Njoku, Pohl, and Martin say we are misleading because AOAA did not inhibit nephrotoxicity. This is nitpicking. Furthermore, nowhere did we claim that AOAA completely inhibited toxicity. AOAA reduced compound A-dependent increases in urine volume, proteinuria and enzymuria. AOAA did not reduce compound A-dependent necrosis and glucosuria. Would it have been preferable for the abstract to have stated “probenecid significantly diminished compound A-induced increases in urine excretion of protein, glucose, and [small alpha, Greek] GST and completely prevented proximal tubular cell necrosis, and AOAA significantly diminished compound A-induced increases in urine volume, protein excretion and [small alpha, Greek] GST excretion but did not significantly alter glucose excretion or the histologic pattern of injury” rather than the summary “probenecid and AOAA diminished compound A nephrotoxicity”? Certainly. More information is always preferable. That is why manuscripts are written. But we were bound by the abstract 250-word limit. We trust that Drs. Njoku, Pohl, and Martin also contend with such word-length restrictions.
Drs. Njoku, Pohl, and Martin misquote our manuscript regarding the aminobenzotriazole experiment. They say we “purport”(a rather charged term) that aminobenzotriazole had no consistent effect on compound A nephrotoxicity, and “we seem to suggest that P450 metabolism of compound A and its metabolites do not have a role in compound A nephrotoxicity.” In fact, aminobenzotriazole decreased glucosuria and necrosis slightly, and had no effect on urine volume, protein and GST excretion. This is not a consistent effect, as we stated. Furthermore, we did not make the conclusion which Drs. Njoku, Pohl and Martin attribute to us. Rather we stated “this does not offer strong evidence to support a substantial role for P450 in rat compound A toxicity.” We stand by that conclusion. Indeed, the objection of Drs. Njoku, Pohl, and Martin is quite puzzling, because Martin et al. also concluded “nor does (compound A) injury require metabolism mediated by cytochrome P450.”
Compound A effects in humans continue to be studied, there exists a “controversy” unfortunately initiated, promulgated, and funded by special interests, and the mechanism of compound A nephrotoxicity in rats remains incompletely elucidated. At present, the preponderance of scientific data indicates a role for the cysteine conjugate [small beta, Greek]-lyase pathway. [1,2,4-10]Formation in vivo of compound A-glutathione conjugates, their cleavage to cysteine conjugates, and metabolism by renal [small beta, Greek]-lyase to reactive intermediates has been demonstrated unambiguously. Inhibition of renal transport by probenecid and of renal [small beta, Greek]-lyase by AOAA diminished histologic and/or biochemical evidence of compound A nephrotoxicity. Protection is not complete, nor is it always, when parent haloalkenes are used with inhibitors. Compound A-glutathione and -cysteine conjugates replicate the nephrotoxicity of compound A, and AOAA partially blocked their toxicity. Finally, the [small alpha, Greek]-methylcysteine analog of the compound A-cysteine conjugate, which is not a substrate for renal [small beta, Greek]-lyase, was not nephrotoxic. Together, these observations suggest involvement of glutathione conjugate formation and S-conjugates transport and metabolism by the S-conjugates/[small beta, Greek]-lyase pathway in compound A nephrotoxicity. Although not apparent, other coexistent mechanisms may also be operant; no claims of exclusivity have been made. Nonetheless, Drs. Njoku, Pohl, and Martin have not offered an alternative hypothesis or supporting data.
Finally, Drs. Njoku, Pohl, and Martin state “maybe it is no longer prudent to rely on reading only abstracts.” It is surprising to learn they had formerly done so.
Evan D. Kharasch, M.D., Ph.D.
Douglas Hankins, M.A.
Research Technologist; Department of Anesthesiology; University of Washington; Seattle, Washington
(Accepted for publication November 13, 1998.)