The nitric oxide prodrug, V-PYRRO/NO, protects against cadmium toxicity and apoptosis at the cellular level

doi:10.1016/j.niox.2005.01.005

Nitric Oxide

Volume 12, Issue 2, March 2005, Pages 114-120

https://doi.org/10.1016/j.niox.2005.01.005 Get rights and content

Abstract

The liver is an important target tissue of cadmium. The compound O²-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2 diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO) prodrug that is metabolized by hepatic P450 enzymes to release NO in hepatocytes. In vivo, V-PYRRO/NO can protect against the toxicity of various hepatotoxicants, including cadmium. Since NO is an effective vasodilator, whether this protective effect against cadmium toxicity is at the level of the hepatic vascular system or actually within the liver cells has not been defined. Thus, we studied the effects of V-PYRRO/NO pretreatment on cadmium-induced toxicity and apoptosis in cultured rat liver epithelial (TRL 1215) cells. Cells were pretreated with V-PYRRO/NO at 500 or 1000 μM for up to 24 h, then exposed to cadmium (as CdCl₂) for additional 24 h and cytotoxicity was measured. Cadmium was significantly less cytotoxic in V-PYRRO/NO (1000 μM) pretreated cells (LC₅₀ = 6.1 ± 0.6 μM) compared to control cells (LC₅₀ = 3.5 ± 0.4 μM). TRL 1215 cells acted upon the prodrug to release NO, producing nitrite levels in the extracellular media after 24 h of exposure to 500 or 1000 μM V-PYRRO/NO measured at 87.0 ± 4.2 and 324 ± 14.8 μM, respectively, compared to basal levels of 7.70 ± 0.46 μM. V-PYRRO/NO alone produced small increases in metallothionein (MT), a metal-binding protein associated with cadmium tolerance. However, V-PYRRO/NO pretreatment greatly enhanced cadmium induction of MT. V-PYRRO/NO pretreatment also markedly reduced apoptotic cell death induced by cadmium (5 μM), apparently by blocking cadmium-induced activation of the c-Jun N-terminal kinase (JNK) pathway. Thus, the prodrug, V-PYRRO/NO, protects against the adverse effects of cadmium directly within rat liver cells apparently through generation of NO and, at least in part, by facilitation of cadmium-induced MT synthesis.

Section snippets

Materials

Cadmium chloride was purchased from Sigma Chemical (St. Louis, MO). V-PYRRO/NO was synthesized as described previously [6]. The structure of V-PYRRO/NO and the mode of NO release have been reported previously [6], [7]. SAPK/JNK assay kits were purchased from New England Biolabs (Beverly, MA).

Cell culture

The TRL 1215 cell line was originally derived from the liver of 10-day-old Fischer F344 rats and cells were cultured as previously described [23]. The cells are diploid and normally nontumorigenic. Cells

V-PYRRO/NO-induced tolerance to cadmium-induced cytotoxicity

TRL 1215 cells were pretreated with V-PYRRO/NO for 24 h. Cells were then incubated with various levels of cadmium for additional 24 h and cytotoxicity was measured. Pretreatment with V-PYRRO/NO significantly reduced cadmium-induced cytotoxicity (Table 1). The LC₅₀ for cadmium in V-PYRRO/NO pretreated cells was 1.7-fold higher than that in control cells. V-PYRRO/NO treatment alone was not cytotoxic.

Nitrite formation after V-PYRRO/NO exposure in TRL 1215 cells

To confirm that V-PYRRO/NO was acted upon by TRL 1215 cells to release NO, cells were incubated with

Discussion

In vivo the NO-releasing prodrug, V-PYRRO/NO delivers NO to the liver in a relatively selective fashion via hepatic metabolism [6]. It has been reported that cultured hepatocytes metabolize V-PYRRO/NO to NO, triggering cyclic guanosine 3′,5′-monophosphate (cGMP) synthesis [6]. V-PYRRO/NO also improves porcine liver hemodynamics after ischemia–reperfusion [5], which likely is due to an effect of NO on the hepatic vasculature. Thus, the protection of the liver from hepatotoxins like

Acknowledgments

The authors thank Drs. Yaxiong Xie and Jun Shen for critical review of this manuscript.

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The reported synthesis of O²-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO), a hepatoprotective agent, was cumbersome and limited by a poor overall yield of 4% from sodium 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO). We report an improved synthesis of V-PYRRO/NO in two steps with a significantly higher overall yield of 40% from PYRRO/NO. Using this protocol, a number of structural analogues of V-PYRRO/NO were prepared in good yields.
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Published by Elsevier Inc.

The nitric oxide prodrug, V-PYRRO/NO, protects against cadmium toxicity and apoptosis at the cellular level

Abstract

Section snippets

Materials

Cell culture

V-PYRRO/NO-induced tolerance to cadmium-induced cytotoxicity

Nitrite formation after V-PYRRO/NO exposure in TRL 1215 cells

Discussion

Acknowledgments

FEBS Lett.

Biochem. Biophys. Res. Commun.

Hepatology

Int. Immunopharmacol.

Toxicol. Lett.

Toxicol. Lett.

Toxicol. Appl. Pharmacol.

Biochem. Biophys. Res. Commun.

Toxicol. Appl. Pharmacol.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Nitric oxide negatively regulates c-Jun N-terminal kinase/stress-activated protein kinase by means of S-nitrosylation

Proc. Natl. Acad. Sci. USA

Progress toward clinical application of the nitric oxide-releasing diazeniumdiolates

Annu. Rev. Pharmacol. Toxicol.

V-PYRRO/NO: an hepato-selective nitric oxide donor improves porcine liver hemodynamics and function after ischemia reperfusion

Transplantation