Polyamine toxicity in mammalian cells in culture
The polyamines, spermidine and spermine, and their diamine precursor putrescine, act as both positive and negative regulators of cell growth. Their oxidation to growth inhibitory aminoaldehydes by a copper-dependent amine oxidase present in ruminant serum was initially thought to be responsible for their toxicity. However, more recently, it has been shown that polyamines are toxic in the absence of ruminant serum and that oxidation by an intracellular amine oxidase may be involved. It is this area of polyamine toxicity that this work has focused on. The model used for a normal fibroblast cell line derived from baby hamster kidney cells (BHK-21/C13). They are routinely grown in Dulbecco's medium supplemented with 10&'37 (v/v) horse serum. There was no extracellular metabolism of spermine in this system. The ID50 value for spermine in BHK cells was 1.03 0.07mM following a 24h exposure to the amine. 2mM-Spermine was cytotoxic, resulting in a significant decrease in cell number, protein content and DNA synthesis after an 8h exposure. Treatment of cells with this concentration of spermine resulted in the accumulation of spermine intracellularly, with levels reaching 11 fold higher than in control cells. Pretreatment of cells with aminoguanidine, an inhibitor of copper-containing amine oxidases, attenuated spermine toxicity, suggesting that oxidation by an intracellular copper-containing amine oxidase plays an important role in spermine toxicity. As well as the copper-dependent amine oxidases, polyamines can also be oxidized by a FAD-dependent peroxisomal amine oxidase. This enzyme is involved in the polyamine retroconversion pathway which converts spermine back to spermidine and putrescine by a series of acetylation and oxidation reactions. Inhibition of this enzyme enhanced the toxicity of spermine, which indicates a protective role for the retroconversion pathway in the removal of the high intracellular spermine pool. This may be linked to enhanced excretion of spermidine and N. 1-acetylspermidine which have been shown to be the main excretory products of BHK cells. Intracellular reduced glutathione levels were depleted in spermine-treated cells. The depletion of glutathione preceded any effect on cell growth and prior depletion of the thiol, by inhibiting its synthesis, enhanced the toxicity of spermine, both suggestive of an important cytoprotective role for glutathione within the cells against spermine toxicity. This may be in the removal of H_2O_2 formed during spermine oxidation, however inactivation of the glutathione redox cycle which is responsible for this did not alter the toxicity of spermine. Glutathione may conjugate with spermine or an oxidative metabolite of spermine, which would act to detoxify the amine and also result in a depletion of intracellular glutathione. When cells that were preloaded with [. 35S]cysteine, were treated with [. 14C]spermine, there was no indication of a double labelled compound in the acid soluble material, indicative of a glutathione spermine conjugate. More studies are required before the formation of such an adduct can be dismissed, as the conjugate may be excreted or broken down, or may be associated with protein thiol groups.