Marilyn L. Cowger

Mechanism of bilirubin toxicity on tissue culture cells: Factors that affect toxicity, reversibility by albumin, and comparison with other respiratory poisons and surfactants

Abstract Various factors that influence the cytotoxic effects of bilirubin, a known electron-transport poison, on mammalian cells in culture have been studied. The cellular uptake of free bilirubin is rapid, being complete in 10 minutes or less following its addition to a cell suspension. Increasing bilirubin concentration increased cell death; there is a lag phase at all concentrations tested, inversely proportional to the concentration of the bile pigment seen with this effect on viability. Cytotoxicity increased at pH 7.6 and below. Albumin-bound bilirubin was nontoxic to tissue culture cells. Albumin added to bilirubin-treated cells resulted in extraction of significant quantities of the cellular-bound bilirubin. However, even when the albumin was added as rapidly as 1 minute after the bilirubin to cells there was still some loss in cell viability. Uncouplers of oxidative phosphorylation and inhibitors of NADH oxidase activity, unlike bilirubin, neither decreased cell viability nor brought about the loss of soluble proteins from cells. Bilirubin more closely resembled agents such as deoxycholate that affect cell membranes in bringing about the loss of cellular proteins. The suggestion is made that the electron-transport lesion induced by bilirubin may not in itself bring about cell death, but that the primary lethal lesion may be on multiple cell membrane systems, perhaps brought about by some alteration of membrane lipids.

Relation of barbiturate structure to DPNH oxidase inhibition

Abstract A series of barbiturates, carbamides, and miscellaneous compounds were examined in order to elucidate the structural requirements for an Amytal-like inhibition of DPNH oxidase. It was found that the essential molecular structure consisted of a nonspecific hydrocarbon group attached to an amide, carbamide, or barbituric acid, these compounds having in common the CONH group. None of the compounds inhibited the oxidation of succinate by succinic oxidase. Similarity was noted between the structural requirements for DPNH oxidase inhibition and for experimental porphyria induction.

Spectral characteristics of bilirubin-bovine albumin complexes

Abstract Spectral characteristics of bilirubin-bovine albumin complexes have been examined. In the presence of excess albumin, the absorption spectrum of the complex did not change from pH 11.5–8.5. As the pH decreased from 8.5 to 5.5, there was a concomitant shift in the absorption maximum from 472 nm to 454 nm, and the spectra intersected at an isobestic point. This transition included the physiologically important pH range and had a pK = 6.6. Between pH 4.5 and pH 3.4 a complex with maximum absorbance at 421 nm was formed and appeared to be dependent on the N-F isomer transformation of albumin. At pH 5.1, there was spectral evidence that 1 mole of albumin binds only 1 mole bilirubin, since difference spectra of bilirubin-albumin solutions containing excess bilirubin were similar to the spectrum of unbound bilirubin. At pH 8.5, 1 mole albumin would appear to bind 1 mole bilirubin with an absorption maximum at 472 nm, although difference spectra indicated that albumin forms additional complexes with an absorption maximum at 424 nm.

Oxidative metabolism of tissue culture cells in the presence of porphyria-inducing drugs☆

The effects of several porphyria-inducing drugs on cultured mammalian cells have been studied. In tissue culture these drugs caused increased glycolysis as indicated by stoichiometric increases in lactic acid production and glucose consumption. Concomitant decreases were noted in ATP levels, catalase activity, and oxygen uptake. Cell multiplication was decreased, while mean cell diameter, RNA, and protein content per cell were increased. The “porphyric” cells took up greater amounts of Fe59, incorporated more Fe59 into cellular hemin and concentrated more total iron per cell. Many of these findings, which are parallel to those observed in the mammalian liver, suggest that oxidative metabolism has been altered by the drugs. Impaired terminal oxidation as the primary lesion in porphyria could explain the multiple areas of metabolism which are affected by porphyria-inducing compounds.

Case reportCongenital erythropoietic porphyria: I. Case report, special studies and therapy☆

Abstract A fourteen year old girl with classic congenital erythropoietic porphyria has recurrent skin bullae, mutilating scarring of the face and hands, hirsutism, porphyrinuria, discolored fluorescent teeth, fluorescent erythroid cells, increased hemolysis and grossly increased excretion of porphyrins. The distribution of discoloration in her primary and secondary teeth suggests that after birth there was a marked decrease in porphyrin production which rose again after the age of one to one and a half years. Two periods of temporary, marked decrease in porphyrinuria following acute minor illness were observed. Bone marrow findings during one of these episodes suggested that they were due to transient erythroid hypoplasia. Intrinsically abnormal red cells are present, with a resultant mild compensated hemolytic process. Less hematologic significant features are hypersplenism and abnormal erythropoiesis. The patients skin lesions seem to be most directly related to increased skin fragility in areas chronically exposed to the sun. Studies of porphyrin excretion indicate that her porphyrin production is about twice what might be expected for a normal child with an equivalent rate of hemoglobin synthesis and that almost all of this excess is of isomer type I, which is incapable of being converted to protoporphyrin and heme. Only a small fraction of her excreted porphyrins can be attributed to those in the peripheral circulation, suggesting that most comes directly from the marrow. The lack of response to therapeutic trials of purine precursors (inosine and adenosine monophosphate) is reported. The therapeutic value of splenectomy, glucocorticoids and hypertransfusion is discussed.

The inhibition of terminal oxidation by porphyrinogenic drugs

Abstract A total of 30 drugs representing a wide variety of molecular species were investigated for their ability: (1) to inhibit reduced nicotinamide adenine dinucleotide oxidase purified from beef heart mitochondria, and (2) to induce increased glycolysis, as measured by lactate production, in cultured mouse fibroblast cells. Most of the drugs tested were chosen because they have been associated with the precipitation of symptoms in clinical cases of porphyria, or have been shown to induce porphyria in an experimental system, or both. The constant relationship between these two properties, namely, the inhibition of electron transport and the exacerbation or induction of porphyria, supports the thesis of a direct metabolic link between impaired terminal oxidation and porphyrinogenesis.

Control of heme biosynthesis: Effect of nicotinamide adenine dinucleotide on the induction of δ-aminolevulinate synthetase☆

Abstract The control of heme biosynthesis in mouse liver was investigated by chemically inducing formation of the rate-limiting enzyme, δ-aminolevulinate synthetase, with either allylisopropylacetamide or dihydrodicarbethoxycollidine. In both instances, the simultaneous administration of nicotinamide adenine dinucleotide depressed enzyme activity. The NAD was shown not to cause enzyme inhibition and was assumed to prevent enzyme induction. Possible modes of action of NAD were considered, especially stimulation of a pathway for NADH oxidation via succinate oxidase, thus bypassing the hypothetical inhibition of NADH oxidase that leads to accelerated heme biosynthesis.

Control of heme biosynthesis: Correlation of porphyrinogenesis with NADH oxidase activity☆

The role of terminal oxidation in the control of heme biosynthesis was investigated. A comparison of the capacity of selected compounds to in-crease porphyrinogenesis in cultured chick embryo liver cells and to inhibit purified beef heart NADH oxidase gave significant correlation co-efficients in two sets of data (r = 0.67, p < 0.01 and r = 0.57, p < 0.05). The influence of similar steric effects and the wide variety of molecular species examined enhanced the significance of the correlation between porphyrin biosynthesis and NADH oxidase activity. Exceptions were found with allylisopropylacetamide and some of the steroids. These exceptions as well as the implications of the correlation data for experimental and clinical porphyrias are discussed.

Liver aldehyde oxidase: Relation to the electron transport systems

Previously, IGO et al. 1 have shown that aldehyde oxidase activity is located in the mitochondrial fraction of pig-liver cells. Aldehyde oxidase purified by the method of MAHLER et al. 2 is similar in several respects to the DPNH dehydrogenases prepared from the particulate electron transport systems of beefand pig-heart mitochondria, and especially the DPNH dehydrogenase described by MACKLER 3. DPNH dehydrogenase and aldehyde oxidase are soluble metalloflavoproteins, and each reacts with oxygen as electron acceptor to form H20 * as a product. In addition, the enzymes are prepared in a similar fashion employing acid-alcohol and heat steps for solubilization. The present paper describes the results of studies which suggest strongly that aldehyde oxidation in liver mitochondria is mediated through the terminal electron transport systems, and that aldehyde oxidase is a solubilized fragment of this system. Mitochondrial suspensions were prepared from rat liver by modifications of the method of SCHNEIDER 4. Aldehyde oxidase was isolated from pig liver as described by M&HLER et al. 2. Difference spectra were recorded using the sensitive split-beam spectrophotometer described by CHANCE 5. Enzymic assays were performed by means of an oxygen polarograph as described previously by IGo AND MACKLER 6. DPNH was obtained from the Sigma Chemical Company, Seconal and Amytal from Eli Lilly and Company, and antimycin A from the Wisconsin Alumni Research Foundation. When acetaldehyde was added to preparations of liver mitochondria the rate of utilization of oxygen was markedly increased as shown in Figs. IA and B. As described previously by several other workers there was a slow rate of oxygen utilization before addition of aldehyde due to the presence of endogenous substrate in the mitochondrial preparations. Addition of ADP to the assay mixture increased the rates of oxygen utilization showing that electron transport was coupled to phosphorylation. The rate of utilization of oxygen by the mitochondrial preparation following acetaldehyde addition was inhibited approx. 80 % by either antimycin A (I ~g ml) or Seconal (I mM) as shown in Figs. IA and B. Amytal (2 mM) produced a similar inhibition. The ability of antimycin and Seconal (or Amytal) to inhibit aldehyde oxidation in mitochondria suggests strongly that the oxidation is mediated through the electron transport systems, since the inhibitors are specific for these systems.