Heme oxygenase (HO) is the rate-limiting enzyme in heme catabois and its activity is induced by many agents, including its substrate heme, heavy metals, UV radiation, and other injurious oxidant conditions. We examined the presence of several regulatory elements in the promoter region of the human HO-1 gene which could possibly account for its induction in response to diverse agents or influences. (11,12). Two HO isozymes, the products of two distinct genes, have been described (13,14). HO-1 is the inducible form which is ubiquitously distributed in mammalian tissues, whereas HO-2 is believed to be constitutively expressed, is not inducible by HO-1 inducers, and is present in tissues such as the brain and testis (14).One of the mechanisms by which hormones, growth factors, and other stimuli induce the expression of genes is by activating various transcription factors. This is a rapid process which frequently involves transcriptional or structural activation of the factor and allows its presence or transfer to the nucleus. These processes may be part of the mechanism by which various agents, including heme, increase HO expression and activity. Previous studies have shown the presence of AP-1-binding sequences and interleukin 6-, metal-, and heat-responsive elements in the HO-1 promoter region and have suggested the involvement of these nuclear factors in the regulation of several genes, including that encoding [13][14][15][16][17][18]. Erythropoietic cells are endowed with HO activity which is inducible by heme (6,7). We therefore used a human-derived erythroleukemic cell line, K562, to examine the presence of transcription factors which might be involved in heme-induced HO-1 expression and to determine whether binding sequences for these factors were present in the promoter region of the human HO-1 gene. tTo whom reprint requests should be addressed. MATERIALS AND METHODS 5987The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

these studies provides direct evidence that the inductive response of human HO to such injurious stimuli represents an important tissue adaptive mechanism for moderating the severity of cell damage produced by these blood components.

Cytochrome P-450-dependent metabolites of arachidonic acid (AA) increased in the kidneys of young, spontaneously hypertensive rats (SHRs) during the period of rapid elevation of blood pressure (BP) but not in adult SHRs or in Wistar Kyoto rats (WKYs) with normal BP. Treatment of SHRs and WKYs with stannous chloride (SnCl2), which selectively depletes renal cytochrome P-450, restored BP to normal, coincident with a natriuresis, in young but not in adult SHRs and did not affect either BP or sodium excretion in WKYs. Depletion of renal cytochrome P-450 was associated with decreased generation of these AA metabolites only in young SHRs. The antihypertensive effect of SnCl2 in young SHRs was greatly reduced by prevention of its cytochrome P-450-depleting action.

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Cytochrome P450 content and activities are increased in the kidneys of spontaneously hypertensive rats (SHR) as compared with those of normotensive, Wistar-Kyoto (WKY), control rats during the period of rapid elevation of blood pressure.

The effects of selected heme analogues on heme oxygenase activity in tissues and on human and rabbit bone marrow hematopoietic colony growth were examined. Zinc protoporphyrin (ZnPP) and zinc mesoporphyrin (ZnMP), at concentrations ranging between 1 and 20 M, produced significant inhibition of human and rabbit bone marrow erythroid (CFU-E, BFU-E) and myeloid (CFU-GM) colony growth. The growth inhibition produced by ZnPP or ZnMP was not overcome with exposure of cultures to elevated levels of the growth factors erythropoietin and granulocytemacrophage colony stimulating factor. In contrast, tin protoporphyrin and tin mesoporphyrin did not display any significant bone marrow toxicity when used at similar concentrations. In other studies, differential effects of tin mesoporphyrin and ZnMP administered intravenously on kidney heme oxygenase were demonstrated. Chromium mesoporphyrin administered intravenously proved lethal to animals. These results indicate that exposure of bone marrow to ZnPP and ZnMP can be deleterious to hematopoietic cells and raise the possibility that ZnPP, which is endogenously formed and found in high concentration in red blood cells in lead-poisoned children, may itself participate in the bone marrow toxicity produced by this metal.Hematopoietic cell growth and differentiation within the bone marrow microenvironment are dependent on a complex interplay of cells, cytokines, growth factors, and heme oxygenase (HO) activity, with the latter enzyme playing a major regulatory role in this system. Heme, a potent inducer of HO expression has been shown to have modulatory effects on hematopoiesis (1). A comprehensive study comparing the effects on hematopoietic cells of synthetic heme analogues, which inhibit HO activity has, not previously been undertaken. Information from this type of study is of special importance because of the clinical potential (2-8) of certain of these compounds.In this study we compared the effects of tin and zinc porphyrins on hematopoietic cell growth and colony formation in animal and human bone marrow cultures. Such cell systems are especially vulnerable to the nature of their microenvironment and thus can provide sensitive indices of the deleterious potential of various chemical agents.The results of this study indicate that zinc porphyrins are toxic to both myeloid and erythroid cell growth even at low concentrations. In contrast, tin porphyrins, even at high concentrations, displayed no toxic effects on hematopoiesis. In other experiments tin and zinc porphyrins were shown to have differing effects on renal HO activity when administered intravenously (i.v.). Chromium mesoporphyrin (CrMP) proved lethal to animals when administered by the iv route.These findings provide additional examples of the differential effects of HO inhibitors on cell functions based on their central metal atom and on their route of administration. The inhibitory actions of zinc porphyrins on bone marrow cell growth represent newly identified deleterious properties of these metalloporphyr...

Three micromethods are described for the assay of enzymes or products of the heme biosynthetic pathway in blood. A fluorometric method for the assay of protoporphyrin may be applied to the rapid screening of children for chronic lead poisoning. A colorimetric assay for δ-aminolevulinic-acid dehydratase may be applied to the detection of acute and chronic lead poisoning. A fluorometric assay for porphyrin formation from prophobilinogen is also described.

Heme oxygenase, the rate-limiting enzyme for heme degradation, can be inhibited by several new synthetic metalloporphyrins. Under certain conditions, a depression in heme oxygenase activity has important clinical significance in the treatment of hyperbilirubinemia, and, in this regard, tin-protoporphyrin has been shown to decrease the production of bilirubin in vitro as well as in vivo. Similarly, our study was concerned with finding a new metalloporphyrin which will inhibit heme oxygenase. Many of the synthetic heme analogs that we analyzed were quite effective inhibitors of heme oxygenase, but the most powerful inhibitor was found to be zinc-deuteroporphyrin IX, 2,4-bisglycol. This metalloporphyrin almost completely inhibits liver heme oxygenase at concentrations as low as 0.5 microM. Its potency as an inhibitor was found to be greater than that of tin-protoporphyrin; the Ki of zinc-deuteroporphyrin IX, 2,4-bisglycol was calculated to be 0.003 microM. In conclusion, we demonstrated that zinc-deuteroporphyrin IX, 2,4-bisglycol has potent inhibitory effects on human liver, kidney and brain heme oxygenase so that this metalloporphyrin can be considered as an alternative to tin-protoporphyrin in the treatment of hyperbilirubinemia.