Protection from doxorubicin-induced cardiac toxicity in mice with a null allele of carbonyl reductase 1.

Doxorubicin is a highly effective antineoplastic agent, but it can produce the serious side effects of acute cardiac injury and chronic congestive heart failure. Carbonyl reductase (CBR) has been implicated in the development of doxorubicin-induced cardiotoxicity. To test whether a decrease in CBR levels was protective against doxorubicin toxicity, we created a null allele of the Cbr1 gene. Mice with one functional copy of the gene (Cbr1 +/-) were healthy and grossly normal despite having decreased levels of Cbr1 transcript and protein. Control and Cbr1 +/- mice were administered doxorubicin at 20 mg/kg i.p. Cbr1 +/- mice showed decreased circulating levels of the cardiotoxic metabolite, doxorubicinol, after administration. Within 2 weeks, 91% of wild-type mice were severely affected (n = 11) compared with 18% of Cbr1 +/- mice (n = 11). Echocardiography and histological analysis showed that Cbr1 +/- mice were protected from gross and cellular level pathologies associated with doxorubicin treatment. Demonstration that inhibition of carbonyl reductase blocks the toxic effects on the heart has important implications for improving the use of doxorubicin in chemotherapy.

Inhibition of superoxide generation from neuronal nitric oxide synthase by heat shock protein 90: implications in NOS regulation.

Besides NO, neuronal NO synthase (nNOS) also produces superoxide (O(2)(-.) at low levels of L-arginine. Recently, heat shock protein 90 (hsp90) was shown to facilitate NO synthesis from eNOS and nNOS. However, the effect of hsp90 on the O(2)(-.) generation from NOS has not been determined yet. The interrelationship between its effects on O(2)(-.) and NO generation from NOS is also unclear. Therefore, we performed electron paramagnetic resonance measurements of O(2)(-.) generation from nNOS to study the effect of hsp90. Purified rat nNOS generated strong O(2)(-.) signals in the absence of L-arginine. In contrast to its effect on NO synthesis, hsp90 dose-dependently inhibited O(2)(-.) generation from nNOS with an IC(50) of 658 nM. This inhibition was not due to O(2)(-.) scavenging because hsp90 did not affect the O(2)(-.) generated by xanthine oxidase. At lower levels of L-arginine where marked O(2)(-.) generation occurred, hsp90 caused a more dramatic enhancement of NO synthesis from nNOS as compared to that under normal L-arginine. Significant O(2)(-.) production was detected from nNOS even at intracellular levels of L-arginine. Adding hsp90 prevented this O(2)(-.) production, leading to enhanced nNOS activity. Thus, these results demonstrated that hsp90 directly inhibited O(2)(-.) generation from nNOS. Inhibition of O(2)(-.) generation may be an important mechanism by which hsp90 enhances NO synthesis from NOS.

Mitochondrial toxin 3-nitropropionic acid induces cardiac and neurotoxicity differentially in mice.

We investigated the effects of 3-nitropropionic acid (3NPA), a previously characterized neurotoxin, in four strains of mice to better understand the molecular basis of variable host responses to this agent. Unexpectedly, we found significant cardiac toxicity that always accompanied the neurotoxicity in all strains of mice in acute and subacute/chronic toxicity testing. Caudate putamen infarction never occurred without cardiac toxicity. All mouse strains tested are sensitive to 3NPA although the C57BL/6 and BALB/c mice require more exposure than 129SVEMS and FVB/n mice. Cardiac toxicity alone was found in 50% of symptomatic mice tested and morphologically, the cardiac toxicity is characterized by diffuse swelling of cardiomyocytes and multifocal coagulative contraction band necrosis. In subacute to chronic exposure, atrial thrombosis, cardiac mineralization, cell loss, and fibrosis are combined with cardiomyocyte swelling and necrosis. Ultrastructurally, mitochondrial swelling occurs initially, followed by disruption of myofilaments. Biochemically, isolated heart mitochondria from the highly sensitive 129SVEMS mice have a significant reduction of succinate dehydrogenase activity, succinate oxygen consumption rates, and heart adenosine triphosphate after 3NPA treatment. The severity of morphological changes parallels the biochemical alterations caused by 3NPA, consistent with cardiac toxicity being a consequence of the effects of 3NPA on succinate dehydrogenase. These experiments show, for the first time, that 3NPA has important cardiotoxic effects as well as neurotoxic effects, and that cardiac toxicity possibly resulting from inhibition of the succinate dehydrogenase in heart mitochondria, contributes to the cause of death in 3NPA poisoning in acute and subacute/chronic studies in mice.

Electron paramagnetic resonance spectroscopy with N-methyl-D-glucamine dithiocarbamate iron complexes distinguishes nitric oxide and nitroxyl anion in a redox-dependent manner: applications in identifying nitrogen monoxide products from nitric oxide synthase.

Though a large number of studies indicate that nitric oxide synthase (NOS) is responsible for NO&z.rad; production in biological systems, controversy still remains concerning whether NOS directly produces NO&z.rad;. Schmidt et al. (PNAS 93:144492, 1996) proposed that NOS first synthesizes nitroxyl anion (NO(-)), which is then converted to NO&z.rad; by superoxide dismutase (SOD). With electron paramagnetic resonance spectroscopy using N-methyl-D-glucamine dithiocarbamate iron (Fe-MGD), we directly detected NO&z.rad; from purified NOS in the absence of SOD (Xia et al., PNAS 94:12705, 1997). We also showed that the requirement for SOD in the previous NO&z.rad; measurements appeared to be due to the high levels of exogenous superoxide production in their reaction system because of the presence of free FAD. However, it was recently questioned whether Fe-MGD can discriminate NO&z.rad; from NO(-) (Komarov et al., FRBM 28:739-742, 2000). In this study we examined the trapping specificity of different redox forms of Fe-MGD. With Fe(2+)-MGD, NO&z.rad; generated characteristic triplet NO&z.rad;-Fe(2+)-MGD signals (g = 2. 04, a(N) = 12.7 G), whereas NO(-) from Angeli's salt was EPR silent. Both NO&z.rad; and NO(-) gave rise to NO&z.rad;-Fe(2+)-MGD signals when Fe(3+)-MGD was used. Strong NO&z.rad; signals were measured from purified nNOS using the NO&z.rad; selective Fe(2+)-MGD and this was not affected by SOD. Thus, spin trapping with Fe-MGD can distinguish NO&z.rad; and NO(-) and this depends on the redox status of the iron. The detection of NO&z.rad; from purified NOS by Fe(2+)-MGD unambiguously reconfirms our previous report that NOS directly synthesizes NO&z.rad; but not NO(-).