Ferrochelatase regulates retinal neovascularization.

Ferrochelatase (FECH) is the terminal enzyme in heme biosynthesis. We previously showed that FECH is required for endothelial cell growth in vitro and choroidal neovascularization in vivo. But FECH has not been explored in retinal neovascularization, which underlies diseases like proliferative diabetic retinopathy and retinopathy of prematurity. Here, we investigated the inhibition of FECH using genetic and chemical approaches in the oxygen-induced retinopathy (OIR) mouse model. In OIR mice, FECH expression is upregulated and co-localized with neovascular tufts. Partial loss-of-function Fechm1Pas  mutant mice showed reduced retinal neovascularization and endothelial cell proliferation in OIR. An intravitreal injection of the FECH inhibitor N-methyl protoporphyrin had similar effects. Griseofulvin is an antifungal drug that inhibits FECH as an off-target effect. Strikingly, intravitreal griseofulvin decreased both pathological tuft formation and areas of vasoobliteration compared to vehicle, suggesting potential as a FECH-targeting therapy. Ocular toxicity studies revealed that intravitreal injection of griseofulvin in adult mice does not disrupt retinal vasculature, function, or morphology. In sum, mutation and chemical inhibition of Fech reduces retinal neovascularization and promotes physiological angiogenesis, suggesting a dual effect on vascular repair upon FECH inhibition, without ocular toxicity. These findings suggest that FECH inhibitors could be repurposed to treat retinal neovascularization.

Complementation studies of the Arabidopsis fc1 mutant substantiate essential functions of ferrochelatase 1 during embryogenesis and salt stress.

Ferrochelatase (FC) is the final enzyme for haem formation in the tetrapyrrole biosynthesis pathway and encoded by two genes in higher plants. FC2 exists predominantly in green tissue, whereas FC1 is constitutively expressed. We intended to substantiate the specific roles of FC1. The embryo-lethal fc1-2 mutant was used to express the two genomic FC-encoding sequences under the FC1 and FC2 promoter and explore the complementation of the FC1 deficiency. Apart from the successful complementation with FC1, expression of FC2 under control of the FC1 promoter (pFC1::FC2) compensates for missing FC1 but not by FC2 promoter expression. The complementing lines pFC1FC2(fc1/fc1) succeeded under standard growth condition but failed under salt stress. The pFC1FC2(fc1/fc1) line exhibited symptoms of leaf senescence, including accelerated loss of haem and chlorophyll and elevated gene expression for chlorophyll catabolism. In contrast, ectopic FC1 expression (p35S::FC1) resulted in increased chlorophyll accumulation. The limited ability of FC2 to complement fc1 is explained by a faster turnover of FC2 mRNA during stress. It is suggested that FC1-produced haem is essential for embryogenesis and stress response. The pFC1::FC2 expression readily complements the fc1-2 embryo lethality, whereas higher FC1 transcript content contributes essentially to stress tolerance.

A cadmium stress-responsive gene AtFC1 confers plant tolerance to cadmium toxicity.

BACKGROUND: Non-essential trance metal such as cadmium (Cd) is toxic to plants. Although some plants have developed elaborate strategies to deal with absorbed Cd through multiple pathways, the regulatory mechanisms behind the Cd tolerance are not fully understood. Ferrochelatase-1 (FC1, EC4.99.1.1) is the terminal enzyme of heme biosynthesis, catalyzing insertion of ferrous ion into protoporphyrin IX. Recent studies have shown that FC1 is involved in several physiological processes. However, its biological function associated with plant abiotic stress response is poorly understood. RESULTS: In this study, we showed that AtFC1 was transcriptionally activated by Cd exposure. AtFC1 overexpression (35S::FC1) lines accumulated more Cd and non-protein thiol compounds than wild-type, and conferred plant tolerance to Cd stress, with improved primary root elongation, biomass and chlorophyll (Chl) content, and low degree of oxidation associated with reduced H2O2, O·2- and peroxides. In contrast, the AtFC1 loss of functional mutant fc1 showed sensitivity to Cd stress. Exogenous provision of heme, the product of AtFC1, partially rescued the Cd-induced toxic phenotype of fc1 mutants by improving the growth of seedlings, generation of glutathione (GSH) and phytochelatins (PCs), and GSH/PCs-synthesized gene expression (e.g. GSH1, GSH2, PCS1, and PCS2). To investigate the mechanism leading to the AtFC1 regulating Cd stress response in Arabidopsis, a transcriptome of fc1 mutant plants under Cd stress was profiled. Our data showed that disfunction of AtFC1 led to 913 genes specifically up-regulated and 522 genes down-regulated in fc1 mutants exposed to Cd. Some of the genes are involved in metal transporters, Cd-induced oxidative stress response, and detoxification. CONCLUSION: These results indicate that AtFC1 would act as a positive regulator of plant tolerance to Cd stress. Our study will broaden our understanding of the role of FC1 in mediating plant response to Cd stress and provide a basis for further exploration of its downstream genes.

Effects of changes in intracellular iron pool on AlkB-dependent and AlkB-independent mechanisms protecting E.coli cells against mutagenic action of alkylating agent.

An Escherichia coli hemH mutant accumulates protoporphyrin IX, causing photosensitivity of cells to visible light. Here, we have shown that intracellular free iron in hemH mutants is double that observed in hemH(+) strain. The aim of this study was to recognize the influence of this increased free iron concentration on AlkB-directed repair of alkylated DNA by analyzing survival and argE3 → Arg(+) reversion induction after λ>320 nm light irradiation and MMS-treatment in E. coli AB1157 hemH and alkB mutants. E.coli AlkB dioxygenase constitutes a direct single-protein repair system using non-hem Fe(II) and cofactors 2-oxoglutarate (2OG) and oxygen (O2) to initiate oxidative dealkylation of DNA/RNA bases. We have established that the frequency of MMS-induced Arg(+) revertants in AB1157 alkB(+)hemH(-)/pMW1 strain was 40 and 26% reduced comparing to the alkB(+)hemH(-) and alkB(+)hemH(+)/pMW1, respectively. It is noteworthy that the effect was observed only when bacteria were irradiated with λ>320 nm light prior MMS-treatment. This finding indicates efficient repair of alkylated DNA in photosensibilized cells in the presence of higher free iron pool and AlkB concentrations. Interestingly, a 31% decrease in the level of Arg(+) reversion was observed in irradiated and MMS-treated hemH(-)alkB(-) cells comparing to the hemH(+)alkB(-) strain. Also, the level of Arg(+) revertants in the irradiated and MMS treated hemH(-) alkB(-) mutant was significantly lower (by 34%) in comparison to the same strain but MMS-treated only. These indicate AlkB-independent repair involving Fe ions and reactive oxygen species. According to our hypothesis it may be caused by non-enzymatic dealkylation of alkylated dNTPs in E. coli cells. In in vitro studies, the absence of AlkB protein in the presence of iron ions allowed etheno(ϵ) dATP and ϵdCTP to spontaneously convert to dAMP and dCMP, respectively. Thus, hemH(-) intra-cellular conditions may favor Fe-dependent dealkylation of modified dNTPs.

Red cells from ferrochelatase-deficient erythropoietic protoporphyria patients are resistant to growth of malarial parasites.

Many red cell polymorphisms are a result of selective pressure by the malarial parasite. Here, we add another red cell disease to the panoply of erythrocytic changes that give rise to resistance to malaria. Erythrocytes from individuals with erythropoietic protoporphyria (EPP) have low levels of the final enzyme in the heme biosynthetic pathway, ferrochelatase. Cells from these patients are resistant to the growth of Plasmodium falciparum malarial parasites. This phenomenon is due to the absence of ferrochelatase and not an accumulation of substrate, as demonstrated by the normal growth of P falciparum parasites in the EPP phenocopy, X-linked dominant protoporphyria, which has elevated substrate, and normal ferrochelatase levels. This observation was replicated in a mouse strain with a hypomorphic mutation in the murine ferrochelatase gene. The parasite enzyme is not essential for parasite growth as Plasmodium berghei parasites carrying a complete deletion of the ferrochelatase gene grow normally in erythrocytes, which confirms previous studies. That ferrochelatase is essential to parasite growth was confirmed by showing that inhibition of ferrochelatase using the specific competitive inhibitor, N-methylprotoporphyrin, produced a potent growth inhibition effect against cultures of P falciparum. This raises the possibility of targeting human ferrochelatase in a host-directed antimalarial strategy.

Increased expression of Fe-chelatase leads to increased metabolic flux into heme and confers protection against photodynamically induced oxidative stress.

Fe-chelatase (FeCh, EC 4.99.1.1) inserts Fe(2+) into protoporphyrin IX (Proto IX) to form heme, which influences the flux through the tetrapyrrole biosynthetic pathway as well as fundamental cellular processes. In transgenic rice (Oryza sativa), the ectopic expression of Bradyrhizobium japonicum FeCh protein in cytosol results in a substantial increase of FeCh activity compared to wild-type (WT) rice and an increasing level of heme. Interestingly, the transgenic rice plants showed resistance to oxidative stress caused not only by the peroxidizing herbicide acifluorfen (AF) as indicated by a reduced formation of leaf necrosis, a lower conductivity, lower malondialdehyde and H2O2 contents as well as sustained Fv/Fm compared to WT plants, but also by norflurazon, paraquat, salt, and polyethylene glycol. Moreover, the transgenic plants responded to AF treatment with markedly increasing FeCh activity. The accompanying increases in heme content and heme oxygenase activity demonstrate that increased heme metabolism attenuates effects of oxidative stress caused by accumulating porphyrins. These findings suggest that increases in heme levels and porphyrin scavenging capacity support a detoxification mechanism serving against porphyrin-induced oxidative stress. This study also implicates heme as possibly being a positive signal in plant stress responses.

Heme A biosynthesis.

Respiration in plants, most animals and many aerobic microbes is dependent on heme A. This is a highly specialized type of heme found as prosthetic group in cytochrome a-containing respiratory oxidases. Heme A differs structurally from heme B (protoheme IX) by the presence of a hydroxyethylfarnesyl group instead of a vinyl side group at the C2 position and a formyl group instead of a methyl side group at position C8 of the porphyrin macrocycle. Heme A synthase catalyzes the formation of the formyl side group and is a poorly understood heme-containing membrane bound atypical monooxygenase. This review presents our current understanding of heme A synthesis at the molecular level in mitochondria and aerobic bacteria. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy.

BACKGROUND: Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-Aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumour margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumour identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues. METHODS: To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyses the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines. RESULTS: Prominent downregulation of FECH mRNA expression was found in glioblastoma tissues compared with normal brain tissues, suggesting that FECH is responsible for PpIX accumulation in glioblastoma cells. Depletion of FECH by small interference RNA enhanced PpIX fluorescence after exposure to 5-ALA concomitant with increased intracellular PpIX accumulation in glioma cells. Silencing of FECH caused marked growth inhibition and apoptosis induction by PDT in glioma cells. CONCLUSION: These results suggest that knockdown of FECH is a potential approach to enhance PpIX fluorescent quality for optimising the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT.

Seasonal palmar keratoderma in erythropoietic protoporphyria indicates autosomal recessive inheritance.

Erythropoietic protoporphyria (EPP) is an inherited disorder that results from partial deficiency of ferrochelatase (FECH). It is characterized clinically by acute photosensitivity and, in 2% of patients, liver disease. Inheritance is usually autosomal dominant with low penetrance but is recessive in about 4% of families. A cross-sectional study of 223 patients with EPP in the United Kingdom identified six individuals with palmar keratoderma. We now show that these and three additional patients, from six families, have an inherited subtype of EPP which is characterized by seasonal palmar keratoderma, relatively low erythrocyte protoporphyrin concentrations, and recessive inheritance. No patient had evidence of liver dysfunction; four patients had neurological abnormalities. Patients were hetero- or homoallelic for nine different FECH mutations; four of which were previously unreported. Prokaryotic expression predicted that FECH activities were 2.7-25% (mean 10.6%) of normal. Neither mutation type nor FECH activity provided an explanation for the unusual phenotype. Our findings show that palmar keratoderma is a clinical indicator of recessive EPP, identify a phenotype that occurs in 38% of reported families with recessive EPP that to our knowledge is previously unreported, and suggest that patients with this phenotype may carry a lower risk of liver disease than other patients with recessive EPP.

Gene dosage analysis identifies large deletions of the FECH gene in 10% of families with erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is an inherited cutaneous porphyria characterized by partial deficiency of ferrochelatase (FECH), accumulation of protoporphyrin IX in erythrocytes, skin, and liver, and acute photosensitivity. Genetic counseling in EPP requires identification of FECH mutations, but current sequencing-based procedures fail to detect mutations in about one in six families. We have used gene dosage analysis by quantitative PCR to identify large deletions of the FECH gene in 19 (58%) of 33 unrelated UK patients with EPP in whom mutations could not be detected by sequencing. Seven deletions were identified, six of which were previously unreported. Breakpoints were identified for six deletions (c.1-7887-IVS1+2425insTTCA; c.1-9629-IVS1+2437; IVS2-1987-IVS4+352del; c.768-IVS7+244del; IVS7+2784-IVS9+108del; IVS6+2350-TGA+95del). Five breakpoints were in intronic repeat sequences (AluSc, AluSq, AluSx, L1MC4). The remaining deletion (Del Ex3-4) is likely to be a large insertion-deletion. Combining quantitative PCR with routine sequencing increased the sensitivity of mutation detection in 189 unrelated UK patients with EPP from 83% (95% CI: 76-87%) to 93% (CI: 88-96%) (P=0.003). Our findings show that large deletions of the FECH gene are an important cause of EPP. Gene dosage analysis should be incorporated into routine procedures for mutation detection in EPP.

Metallation of the transition-state inhibitor N-methyl mesoporphyrin by ferrochelatase: implications for the catalytic reaction mechanism.

Insertion of metals into various tetrapyrroles is catalysed by a group of enzymes called chelatases, e.g. nickel, cobalt, magnesium and ferro-chelatase. It has been proposed that catalytic metallation includes distorting the porphyrin substrate by the enzyme towards a transition state-like geometry in which at least one of the pyrrole rings will be available for metal chelation. Here, we present a study of metal insertion into the transition-state inhibitor of protoporphyrin IX ferrochelatase, N-methyl mesoporphyrin (N-MeMP), by time-resolved crystallography and mass spectrometry with and without the presence of ferrochelatase. The results show that metallation of N-MeMP has a very limited effect on the conformation of the residues that participate in porphyrin and metal binding. These findings support theoretical data, which indicate that product release is controlled largely by the strain created by metal insertion into the distorted porphyrin. The results suggest that, similar to non-catalytic metallation of N-MeMP, the ferrochelatase-assisted metallation depends on the ligand exchange rate for the respective metal. Moreover, ferrochelatase catalyses insertion of Cu(II) and Zn(II) into N-MeMP with a rate that is about 20 times faster than non-enzymatic metallation in solution, suggesting that the catalytic strategy of ferrochelatase includes a stage of acceleration of the rate of ligand exchange for the metal substrate. The greater efficiency of N-MeMP metallation by Cu(II), as compared to Zn(II), contrasts with the K(m) values for Zn(II) (17 microM) and Cu(II) (170 microM) obtained for metallation of protoporphyrin IX. We suggest that this difference in metal specificity depends on the type of distortion imposed by the enzyme on protoporphyrin IX, which is different from the intrinsic non-planar distortion of N-MeMP. A mechanism of control of metal specificity by porphyrin distortion may be general for different chelatases, and may have common features with the mechanism of metal specificity in crown ethers.

Photosystem II assembly in CP47 mutant of Synechocystis sp. PCC 6803 is dependent on the level of chlorophyll precursors regulated by ferrochelatase.

Accumulation of chlorophyll and expression of the chlorophyll (Chl)-binding CP47 protein that serves as the core antenna of photosystem II are indispensable for the assembly of a functional photosystem II. We have characterized the CP47 mutant with an impaired photosystem II assembly and its two spontaneous pseudorevertants with their much improved photoautotrophic growth. The complementing mutations in these pseudorevertants were previously mapped to the ferrochelatase gene (1). We demonstrated that complementing mutations dramatically decrease ferrochelatase activity in pseudorevertants and that this decrease is responsible for their improved photoautotrophic growth. Photoautotrophic growth of the CP47 mutant was also restored by in vivo inhibition of ferrochelatase by a specific inhibitor. The decrease in ferrochelatase activity in pseudorevertants was followed by increased steady-state levels of Chl precursors and Chl, leading to CP47 accumulation and photosystem II assembly. Similarly, supplementation of the CP47 mutant with the Chl precursor Mg-protoporphyrin IX increased the number of active photosystem-II centers, suggesting that synthesis of the mutated CP47 protein is enhanced by an increased Chl availability in the cell. The probable role of ferrochelatase in the regulation of Chl biosynthesis is discussed.

Involvement of ABC7 in the biosynthesis of heme in erythroid cells: interaction of ABC7 with ferrochelatase.

A mitochondrial half-type ATP-binding cassette (ABC) protein, ABC7, plays a role in iron homeostasis in mitochondria, and defects in human ABC7 were shown to be responsible for the inherited disease X-linked sideroblastic anemia/ataxia. We examined the role of ABC7 in the biosynthesis of heme in erythroid cells where hemoglobin is a major product of iron-containing compounds. RNA blots showed that the amount of ABC7 mRNA in dimethylsulfoxide (Me(2)SO)-treated mouse erythroleukemia (MEL) cells increased markedly in parallel with the induction of the mRNA expression of ferrochelatase, the last enzyme in the pathway to synthesize heme. The transfection of the antisense oligonucleotide to mouse ABC7 mRNA into Me(2)SO-treated MEL cells led to a decrease of heme production, as compared with sense oligonucleotide-transfected cells. ABC7 protein was shown to be colocalized with ferrochelatase in mitochondria, as assessed by immunostaining. Furthermore, in vitro and in vivo pull-down assays revealed that ABC7 protein is interacted with the carboxy-terminal region containing the iron-sulfur cluster of ferrochelatase. The transient expression of ABC7 in mouse embryo liver BNL-CL2 cells resulted in an increase in the activity and level of ferrochelatase and thioredoxin, a cytosolic protein containing iron-sulfur. These increases were also observed in MEL cells stably expressing ABC7. When ABC7 transfectants were treated with Me(2)SO, an increase in cellular heme concomitant with a marked induction of the expression of ferrochelatase was observed. The extent of these increases was 3-fold greater than in control cells. The results indicated that ABC7 positively regulates not only the expression of extramitochondrial thioredoxin but also that of an intramitochondrial iron-sulfur-containing protein, ferrochelatase. Then, the expression of ABC7 contributes to the production of heme during the differentiation of erythroid cells.

In vivo and in vitro studies of Bacillus subtilis ferrochelatase mutants suggest substrate channeling in the heme biosynthesis pathway.

Ferrochelatase (EC 4.99.1.1) catalyzes the last reaction in the heme biosynthetic pathway. The enzyme was studied in the bacterium Bacillus subtilis, for which the ferrochelatase three-dimensional structure is known. Two conserved amino acid residues, S54 and Q63, were changed to alanine by site-directed mutagenesis in order to detect any function they might have. The effects of these changes were studied in vivo and in vitro. S54 and Q63 are both located at helix alpha3. The functional group of S54 points out from the enzyme, while Q63 is located in the interior of the structure. None of these residues interact with any other amino acid residues in the ferrochelatase and their function is not understood from the three-dimensional structure. The exchange S54A, but not Q63A, reduced the growth rate of B. subtilis and resulted in the accumulation of coproporphyrin III in the growth medium. This was in contrast to the in vitro activity measurements with the purified enzymes. The ferrochelatase with the exchange S54A was as active as wild-type ferrochelatase, whereas the exchange Q63A caused a 16-fold reduction in V(max). The function of Q63 remains unclear, but it is suggested that S54 is involved in substrate reception or delivery of the enzymatic product.

The structure of Saccharomyces cerevisiae Met8p, a bifunctional dehydrogenase and ferrochelatase.

Sirohaem is a tetrapyrrole-derived prosthetic group that is required for the essential assimilation of sulfur and nitrogen into all living systems as part of the sulfite and nitrite reductase systems. The final two steps in the biosynthesis of sirohaem involve a beta-NAD(+)-dependent dehydrogenation of precorrin-2 to generate sirohydrochlorin followed by ferrochelation to yield sirohaem. In Saccharomyces cerevisiae, Met8p is a bifunctional enzyme that carries out both of these reactions. Here, we report the 2.2 A resolution crystal structure of Met8p, which adopts a novel fold that bears no resemblance to the previously determined structures of cobalt- or ferro-chelatases. Analysis of mutant proteins suggests that both catalytic activities share a single active site, and that Asp141 plays an essential role in both dehydrogenase and chelatase processes.

The penetrance of dominant erythropoietic protoporphyria is modulated by expression of wildtype FECH.

Erythropoietic protoporphyria (EPP) is an inherited disorder of heme biosynthesis caused by a partial deficiency of ferrochelatase (FECH, EC 4.99.1.1). EPP is transmitted as an autosomal dominant disorder with an incomplete penetrance. Using haplotype segregation analysis, we have identified an intronic single nucleotide polymorphism (SNP), IVS3-48T/C, that modulates the use of a constitutive aberrant acceptor splice site. The aberrantly spliced mRNA is degraded by a nonsense-mediated decay mechanism (NMD), producing a decreased steady-state level of mRNA and the additional FECH enzyme deficiency necessary for EPP phenotypic expression.

Ferrochelatase is present in Brucella abortus and is critical for its intracellular survival and virulence.

Brucella spp. are pathogenic bacteria that cause brucellosis, an animal disease which can also affect humans. Although understanding the pathogenesis is important for the health of animals and humans, little is known about virulence factors associated with it. In order for chronic disease to be established, Brucella spp. have developed the ability to survive inside phagocytes by evading cell defenses. It hides inside vacuoles, where it then replicates, indicating that it has an active metabolism. The purpose of this work was to obtain better insight into the intracellular metabolism of Brucella abortus. During a B. abortus genomic sequencing project, a clone coding a putative gene homologous to hemH was identified and sequenced. The amino acid sequence revealed high homology to members of the ferrochelatase family. A knockout mutant displayed auxotrophy for hemin, defective intracellular survival inside J774 and HeLa cells, and lack of virulence in BALB/c mice. This phenotype was overcome by complementing the mutant strain with a plasmid harboring wild-type hemH. These data demonstrate that B. abortus synthesizes its own heme and also has the ability to use an external source of heme; however, inside cells, there is not enough available heme to support its intracellular metabolism. It is concluded that ferrochelatase is essential for the multiplication and intracellular survival of B. abortus and thus for the establishment of chronic disease as well.

Structure and function of ferrochelatase.

Ferrochelatase is the terminal enzyme of the heme biosynthetic pathway in all cells. It catalyzes the insertion of ferrous iron into protoporphyrin IX, yielding heme. In eukaryotic cells, ferrochelatase is a mitochondrial inner membrane-associated protein with the active site facing the matrix. Decreased values of ferrochelatase activity in all tissues are a characteristic of patients with protoporphyria. Point-mutations in the ferrochelatase gene have been recently found to be associated with certain cases of erythropoietic protoporphyria. During the past four years, there have been considerable advances in different aspects related to structure and function of ferrochelatase. Genomic and cDNA clones for bacteria, yeast, barley, mouse, and human ferrochelatase have been isolated and sequenced. Functional expression of yeast ferrochelatase in yeast strains deficient in this enzyme, and expression in Escherichia coli and in baculovirus-infected insect cells of different ferrochelatase cDNAs have been accomplished. A recently identified (2Fe-2S) cluster appears to be a structural feature shared among mammalian ferrochelatases. Finally, functional studies of ferrochelatase site-directed mutants, in which key amino acids were replaced with residues identified in some cases of protoporphyria, will be summarized in the context of protein structure.

Porphyrin synthesis by murine epidermal cells.

Incubation of murine epidermal cells with delta-aminolevulinic acid (ALA) resulted in a dose- and time-dependent accumulation of porphyrins, predominantly of protoporphyrin. Porphyrin accumulation decreased in the presence of iron, and the iron-mediated decrease was partially reversed by CaMg EDTA (1.25-10.0 mM), suggesting that there is functionally active ferrochelatase in these cells. This study suggests that these cells may be a useful model for the study of cutaneous porphyrin metabolism involving ferrochelatase activity.