César F. Polo

Reducing Sugars Trigger δ-Aminolevulinic Dehydratase Inactivation: Evidence of In Vitro Aspirin Prevention

1. The effect of in vitro glycation on delta-aminolevulinic dehydratase (ALA-D) under several experimental conditions was investigated. When preincubated with 500 mM glucose at 37 degrees C for 20 hr, ALA-D was 80% inactivated and glycated hemoglobin levels were increased more than fourfold. 2. Thiobarbituric acid species were not modified during glycation; therefore ALA-D inactivation cannot be attributed to glucose autoxidation. 3. Acetyl salicylic acid was effective in preventing both hemoglobin glycation and ALA-D inactivation by glucose. 4. A method has been developed for measuring protein glycation in vitro, in a crude preparation of red blood cells, which can also be applied to sugars other than glucose.

STZ-induced diabetes in mice and heme pathway enzymes. Effect of allylisopropylacetamide and α-tocopherol

A frequent coexistence of diabetes and porphyria disease has been reported. Under normal conditions, porphyrin biosynthesis is well regulated to only form the amount of heme required for the synthesis of the various hemoproteins. The activity of some heme enzymes and rhodanese in streptozotocin (STZ) induced diabetic mice and in allylisopropylacetamide (AIA) induced experimental acute porphyria mice has been examined. The role of alpha-tocopherol (alpha-T), reported to prevent protein glycation in vitro, has also been investigated. AIA induced hepatic delta-aminolevulinic acid synthetase (ALA-S) activity in control animals but was ineffective in the diabetic group. alpha-Tocopherol did not modify ALA-S activity in either group. delta-Aminolevulinic acid dehydratase (ALA-D) and deaminase activities were significantly diminished both in liver and blood of diabetic animals. alpha-Tocopherol prevented inhibition of ALA-D, deaminase and blood rhodanese activities in diabetic animals but alpha-tocopherol by itself did not affect the basal levels of the enzymes studied. The potential use of alpha-tocopherol to prevent late complications of diabetes, including the onset of a porphyria like syndrome is considered.

Heme regulation in mouse mammary carcinoma and liver of tumor bearing mice-I. Effect of allyl-isopropylacetamide and veronal on δ -aminolevulinate synthetase, cytochrome P-450 and cytochrome oxidase

1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of delta-aminolevulinate synthetase (ALA-S), cytochrome P-450 (cyt P-450) and cytochrome oxidase were determined in tumor (T) and liver of both normal mice (NM) and T bearing mice (TBM). 2. Basal levels of ALA-S were nearly the same in either source. The amount of cyt P-450 was lower in TBM liver than in NM liver, and no detectable in T. While the basal activity of cytochrome oxidase in TBM liver and T were higher than those of NM liver. 3. In AIA intoxicated animals there was a lower induction of ALA-S in liver of TBM than in NM liver. There was no induction in T ALA-S. The loss of cyt P-450 was less in TBM liver when compared with NM liver. 4. The induction level of cyt P-450 after veronal administration was nearly the same in liver of both TBM and NM. 5. We conclude that lower induction of liver ALA-S activity in TBM liver is due to correspondingly lower drug metabolism ability of TBM liver. Otherwise our results suggest that the control mechanism operating in T and probably in its original tissue are different from those described for normal liver.

Regulation of heme pathway in regenerating mouse liver

1. delta-Aminolevulinic acid synthetase (ALA-S), rhodanese and microsomal heme oxygenase (MHO), were quantitated in Cl4C induced regenerating mouse liver. 2. Maximal hepatomegalia was observed at 48 hr after i.p. injection of a single dose of the toxin. 3. ALA-S activity decreased on day 2, and then significantly increased (50%) between days 3 and 7, returning afterwards to control values. 4. Cytoplasmic rhodanese, as well as MHO activities, exhibited a clear correlation as compared with the ALA-S activity profile. 5. Porphyrin biosynthesis from precursor delta-aminolevulinic acid (ALA) was significantly increased even after 15 days of intoxication. 6. Present results would indicate that Cl4C is acting in a dual fashion.

Altered Heme Pathway Regulation and Drug Metabolizing Enzyme System in a Mouse Model of Hepatocarcinogenesis: Effect of Veronal

1. Male CF 1 mice were fed p-dimethylaminoazobenzene (DAB) for 35 days and received 5,5-diethylbarbituric acid, before or after DAB treatment, with the purpose of investigating whether the onset of the preinitiation stage of carcinogenesis alters the natural regulatory mechanism of the heme pathway. 2. Changes detected in drug metabolizing enzymes are likely to be the consequence of a primary deregulation mechanism of heme metabolism, shown by an increase in delta-aminolevulinic acid synthetase activity and a decrease in microsomal heme oxygenase, which would finally lead to a great enhancement of cytochrome P450 levels. 3. The alterations found here would give rise to a pattern distinctive to that usually observed in the so-called resistant hepatocyte.

Heme biosynthesis pathway regulation in a model of hepatocarcinogenesis pre-initiation

1. Heme regulation before the appearance of hyperplastic nodules was investigated in mice models of hepatocarcinogenesis. 2. With this aim 5-aminolaevulinate synthetase (ALA-S), microsomal heme-oxygenase (MHO), mitochondrial and cytoplasmic rhodanese activities were examined throughout a period of 35 days in animals exposed to dietary p-dimethylaminoazobenzene (DAB). 3. ALA-S activity was significantly diminished (50%) on day 14, then showing a sharply rising profile from day 28 onwards, and reaching 350% on day 35. 4. A similar profile was observed for mitochondrial rhodanese activity. 5. Changes in MHO and cytoplasmic rhodanese activities were almost the opposite to those observed for ALA-S. 6. The distinctive alteration in mitochondrial and cytoplasmic rhodanese would suggest that it plays a subtle role in ALA-S regulation during carcinogenesis initiation through a mechanism that appears to involve subcellular localization controls perhaps by means of the breakage of cystine trisulphide postulated to act as an ALA-S activator. 7. Taking into account the present results, we suggest a probable mechanism for the onset of hepatocarcinogenesis that includes a primary activating liver status, provoking biochemical aberration leading to the stage of initiation of hepatocarcinogenesis involving the whole organ.

Rhodanese and ALA-S in mammary tumor and liver from normal and tumor-bearing mice

1. Basal levels and allyl-isopropylacetamide (AIA) or veronal induced levels of delta-amino-levulinate synthetase (ALA-S), cytoplasmic and mitochondrial rhodanese were determined in tumor (T) and liver of both normal mice (NM) and T-bearing mice (TBM). 2. Rhodanese tumoral mitochondrial levels were higher than the hepatic normal mitochondrial fraction, while the cytoplasmic activity was nearly equal in all sources. 3. In neither case was the activity of tumoral ALA-S and rhodanese altered by any of the porphyrinogenic drugs. 4. Mitochondrial and cytoplasmic rhodanese activity was also measured in tumor and liver of TBM at different intervals after transplantation. We concluded that the behaviour of rhodanese is a property inherent to the tissue and not one attained with time.

The effect of griseofulvin on the heme pathway-II. An exhaustive analysis during short and long-term challenge

1. A clear biphasic response of the enzyme activities as a function of intoxication time due to the topical cutaneous griseofulvin treatment was observed. 2. The initial acute induction of ALA-S activity would be due to depletion of free heme in the regulatory pool caused by cytochrome P 450 destruction. 3. The second induction peak, would be due to less heme formation, secondary to the ferrochelatase inhibition, as expected for the erythropoietic protoporphyria model. 4. The biphasic response of hepatic ALA-D and PBGase activities would be related to ALA-S activity changes and the subsequent augmented available substrates. 5. Endogenous liver porphyrin distribution in cytosolic, mitochondrial and nuclear fractions was investigated. 6. The in vitro biosynthesis of porphyrins confirmed both the biphasic model and the hepatic porphyrins subcellular distribution. 7. Two mechanisms to explain the action of griseofulvin at shorter and longer times of intoxication are proposed.