Bruce C. Frykholm

Succinylacetone, a potent inhibitor of heme biosynthesis: Effect on cell growth, heme content and δ-aminolevulinic acid dehydratase activity of malignant murine erythroleukemia cells

Abstract 4,6-Dioxoheptanoic acid (succinylacetone, SA) was examined with regard to its ability to a) inhibit the second enzyme of the heme pathway, δ-aminolevulinic acid (ALA) dehydratase, b) lower the heme concentration, and c) inhibit cell growth of murine erythroleukemia (MEL) cells in culture. SA profoundly inhibited ALA dehydratase in broken cell preparations at concentrations as low as 10−7 M. The stimulation of hemoglobin production by DMSO and butyrate in MEL cells was inhibited by the addition of SA to the cell medium. When 1 mM SA was added to the medium, there was a profound inhibition of ALA dehydratase activity, and the heme concentration of cells declined progressively with each cell division. Cell growth was markedly inhibited after two cell divisions.

Hematin therapy for acute porphyria.

1. A therapeutic trial of intravenous hematin is presented. Eleven cases of AIP and one of VP who did not improve with conventional treatment (high carbohydrate intake) received this new agent. 2. Urinary ALA, PBG and, when possible, uroporphyrin and coproporphyrin were used to monitor the chemical response to the treatment. Objective clinical parameters of hypertension and tachycardia were followed when present in addition to subjective estimates of acute porphyric symptomatology (abdominal pain, backache, extremity pain and paresthesias, weakness, depression, etc.). 3. At a dosage of approximately 3 mg/kg, diminution of urinary ALA and PBG excretion was achieved in every patients. Hypertension and tachycardia improved in those instances where they were observed in association with the attack. Also, subjective improvements in the clinical status of the patients were observed frequently. 4. Hematin appears to be a promising therapeutic agent for the treatment of acute attack forms of porphyria.

Neurotoxic aspects of porphyrinopathies: Lead and succinylacetone

Abstract Neurotoxic effects of heavy metals and polyhalogenated hydrocarbons frequently occur at low levels of exposure, in some cases below those levels where direct toxic actions of these compounds have been demonstrated. Since alterations in heme synthesis are effects common to many of these compounds, the potential role of altered heme synthesis in producing or contributing to their neurotoxicity was studied. Rats with acute and chronic lead exposure were compared to rats whose heme synthesis was inhibited by succinylacetone, as a semichronic model of the hereditary heme synthesis disorder, acute intermittent porphyria. Both treatments produce significant inhibition in activity of the enzyme δ-aminolevulinic acid dehydrase and elevations in the heme precursor δ-aminolevulinic acid (ALA) in tissues and urine. Associated with increased ALA is a significant inhibition of neurotransmission utilizing the amino acid γ-aminobutyric acid (GABA), expressed chemically and behaviorally. The results suggest that in addition to their direct molecular neurotoxicity, porphyrinopathic compounds such as lead may, through altering heme synthesis, adversely affect the brain at low levels of exposure.

Effect of heme depletion on growth, protein synthesis and respiration of murine erythroleukemia cells

Abstract 4,6-Dioxoheptanoic acid (succinylacetone, SA) profoundly inhibits δ-aminolevulinic acid (ALA) dehydratase, the second enzyme of the heme biosynthetic pathway. When murine eyrthroleukemia (MEL) cells were grown in defined medium containing 0.3–1.0 mM SA and gelatin as the protein source, cellular heme concentrations decreased markedly and growth was inhibited. Growth inhibition was partially reversed when hematin (300 pmoles/ml) was added with SA on day 0, but no reversal of growth inhibition was seen when this concentration of hematin was added on day 4. Complete reversal of growth inhibition produced by 0.3 mM SA occurred when hematin was added in concentrations of 1–2 nmoles/ml on day 0. Cellular heme content and growth rates indicate that exogenously added hematin was taken up from defined medium containing only gelatin as the protein component. The decrease of cellular heme concentration was accompanied by a decrease of protein synthesis, which, like growth inhibition by SA, was partially reversed by addition of hematin to the medium. Although no significant impairment of endogenous cellular respiration was observed in these studies, there was a decrease in NADH-stimulated oxygen consumption in heme-depleted cells, suggesting diminished respiratory reserve in these cells.

Growth inhibitory activity of succinylacetone: studies with Walker 256 carcinosarcoma, Novikoff hepatoma and L1210 leukemia.

Succinylacetone (SA, 4,6-dioxoheptanoic acid) inhibits d-aminolevulinic acid dehydrase, the second enzyme of the heme biosynthetic pathway and thereby inhibits heme biosynthesis. In the present study SA is shown to inhibit the growth of the Walker carcinosarcoma (W256) in vitro and in vivo, the Novikoff hepatoma in vivo, and L1210 leukemia in vitro, but only slightly in vivo. Rats can tolerate significantly larger doses of SA for at least twice as long as were administered in the present study without gross evidence of toxicity. In contrast to findings in previously published studies with murine erythroleukemia cells, the inhibition of growth of L1210 and W256 cells by SA in vitro is not accompanied by a decrease in cellular heme and is not reversed by addition of hematin to the medium. This suggests a second mechanism of growth inhibition of tumor cells unrelated to heme biosynthesis. Although the growth of both W256 and L1210 cells was markedly inhibited by the same concentration of SA in culture, there was a great difference in responsiveness in vivo, in that much greater inhibition of the growth of the Walker tumor was produced by SA.