Characterizing control of memory CD8 T cell differentiation by BTB-ZF transcription factor Zbtb20.

Members of the BTB-ZF transcription factor family regulate the immune system. Our laboratory identified that family member Zbtb20 contributes to the differentiation, recall responses, and metabolism of CD8 T cells. Here, we report a characterization of the transcriptional and epigenetic signatures controlled by Zbtb20 at single-cell resolution during the effector and memory phases of the CD8 T cell response. Without Zbtb20, transcriptional programs associated with memory CD8 T cell formation were up-regulated throughout the CD8 T response. A signature of open chromatin was associated with genes controlling T cell activation, consistent with the known impact on differentiation. In addition, memory CD8 T cells lacking Zbtb20 were characterized by open chromatin regions with overrepresentation of AP-1 transcription factor motifs and elevated RNA- and protein-level expressions of the corresponding AP-1 components. Finally, we describe motifs and genomic annotations from the DNA targets of Zbtb20 in CD8 T cells identified by cleavage under targets and release under nuclease (CUT&RUN). Together, these data establish the transcriptional and epigenetic networks contributing to the control of CD8 T cell responses by Zbtb20.

Plasma DNA as a "liquid biopsy" incompletely complements tumor biopsy for identification of mutations in a case series of four patients with oligometastatic breast cancer.

PURPOSE: Circulating tumor DNA in plasma may present a minimally invasive opportunity to identify tumor-derived mutations to inform selection of targeted therapies for individual patients, particularly in cases of oligometastatic disease where biopsy of multiple tumors is impractical. To assess the utility of plasma DNA as a "liquid biopsy" for precision oncology, we tested whether sequencing of plasma DNA is a reliable surrogate for sequencing of tumor DNA to identify targetable genetic alterations. METHODS: Blood and biopsies of 1-3 tumors were obtained from 4 evaluable patients with advanced breast cancer. One patient provided samples from an additional 7 tumors post-mortem. DNA extracted from plasma, tumor tissues, and buffy coat of blood were used for probe-directed capture of all exons in 149 cancer-related genes and massively parallel sequencing. Somatic mutations in DNA from plasma and tumors were identified by comparison to buffy coat DNA. RESULTS: Sequencing of plasma DNA identified 27.94 ± 11.81% (mean ± SD) of mutations detected in a tumor(s) from the same patient; such mutations tended to be present at high allelic frequency. The majority of mutations found in plasma DNA were not found in tumor samples. Mutations were also found in plasma that matched clinically undetectable tumors found post-mortem. CONCLUSIONS: The incomplete overlap of genetic alteration profiles of plasma and tumors warrants caution in the sole reliance of plasma DNA to identify therapeutically targetable alterations in patients and indicates that analysis of plasma DNA complements, but does not replace, tumor DNA profiling. TRIAL REGISTRATION: Subjects were prospectively enrolled in trial NCT01836640 (registered April 22, 2013).

Obesity and fatty liver are prevented by inhibition of the aryl hydrocarbon receptor in both female and male mice.

Inhibition of the aryl hydrocarbon receptor (AHR) prevents Western diet-induced obesity and fatty liver in C57Bl/6J (B6) male mice. The AHR is a ligand-activated nuclear receptor that regulates genes involved in xenobiotic metabolism and T-cell differentiation. Here, we tested the hypothesis that AHR antagonism would also prevent obesity and fatty liver in female mice and that B6 mice (higher-affinity AHR) and congenic B6.D2 mice (lower-affinity AHR) would differentially respond to AHR inhibition. Female and male adult B6 and B6.D2 mice were fed control and Western diets with and without α-naphthoflavone (NF), an AHR inhibitor. A nonlinear mixed-model analysis was developed to project asymptote body mass. We found that obesity, adiposity, and liver steatosis were reduced to near control levels in all female and male B6 and B6.D2 experimental groups fed Western diet with NF. However, differences were noted in that female B6.D2 vs B6 mice on Western diet became more obese; and in general, female mice compared with male mice had a greater fat mass to body mass ratio, were less responsive to NF, and had reduced liver steatosis and hepatomegaly. We report that male mice fed Western diet containing NF or CH-223191, another AHR inhibitor, caused reduced mRNA levels of several liver genes involved in metabolism, including Cyp1b1 and Scd1, offering evidence for a possible mechanism by which the AHR regulates obesity. In conclusion, although there are some sex- and Ahr allelic-dependent differences, AHR inhibition prevents obesity and liver steatosis in both males and females regardless of the ligand-binding capacity of the AHR. We also present evidence consistent with the notion that an AHR-CYP1B1-SCD1 axis is involved in obesity, providing potentially convenient and effective targets for treatment.

Inhibition of the aryl hydrocarbon receptor prevents Western diet-induced obesity. Model for AHR activation by kynurenine via oxidized-LDL, TLR2/4, TGFß, and IDO1.

Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor ß1 (TGFß1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFß1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR with its broad ligand binding specificity is a promising candidate for a potentially simple therapeutic approach for the prevention and treatment of obesity and associated complications.

SMAD4-dependent polysome RNA recruitment in human pancreatic cancer cells.

Pancreatic cancer is the fourth leading cause of cancer death in the United States because most patients are diagnosed too late in the course of the disease to be treated effectively. Thus, there is a pressing need to more clearly understand how gene expression is regulated in cancer cells and to identify new biomarkers and therapeutic targets. Translational regulation is thought to occur primarily through non-SMAD directed signaling pathways. We tested the hypothesis that SMAD4-dependent signaling does play a role in the regulation of mRNA entry into polysomes and that novel candidate genes in pancreatic cancer could be identified using polysome RNA from the human pancreatic cancer cell line BxPC3 with or without a functional SMAD4 gene. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for most of the remaining polysome RNA, levels are regulated via a "tagging" of the RNAs in the nucleus for rapid entry into the polysomes; (iv) a SMAD4-dependent pathway appears to indeed play a role in regulating mRNA entry into polysomes; and (v) a gene list derived from differentially expressed polysome RNA in BxPC3 cells generated new candidate genes and cell pathways potentially related to pancreatic cancer.

Obesity is mediated by differential aryl hydrocarbon receptor signaling in mice fed a Western diet.

BACKGROUND: Obesity is a growing worldwide problem with genetic and environmental causes, and it is an underlying basis for many diseases. Studies have shown that the toxicant-activated aryl hydrocarbon receptor (AHR) may disrupt fat metabolism and contribute to obesity. The AHR is a nuclear receptor/transcription factor that is best known for responding to environmental toxicant exposures to induce a battery of xenobiotic-metabolizing genes. OBJECTIVES: The intent of the work reported here was to test more directly the role of the AHR in obesity and fat metabolism in lieu of exogenous toxicants. METHODS: We used two congenic mouse models that differ at the Ahr gene and encode AHRs with a 10-fold difference in signaling activity. The two mouse strains were fed either a low-fat (regular) diet or a high-fat (Western) diet. RESULTS: The Western diet differentially affected body size, body fat:body mass ratios, liver size and liver metabolism, and liver mRNA and miRNA profiles. The regular diet had no significant differential effects. CONCLUSIONS: The results suggest that the AHR plays a large and broad role in obesity and associated complications, and importantly, may provide a simple and effective therapeutic strategy to combat obesity, heart disease, and other obesity-associated illnesses.

Arsenic decreases RXRα-dependent transcription of CYP3A and suppresses immune regulators in hepatocytes.

Arsenite is critical pharmacologically as a treatment for advanced stage blood cancer. However, environmental exposure to arsenic results in multiple diseases. Previous studies have shown that arsenic decreases expression of CYP3A, a critical drug metabolizing enzyme in human and rat liver. In addition, acute and chronic arsenic exposure in liver stimulates an inflammatory response. Our work has shown that arsenite decreases nuclear levels of RXRα the nuclear receptor that, as a heterodimer partner with PXR, transactivates the CYP3A gene. These results suggest that arsenite decreases transcription of CYP3A by decreasing RXRα. The present report shows that exposure to 5 µM arsenite decreased the activity of a rat CYP3A promoter luciferase reporter in HepG2 cells. The activity of a RARE-luciferase reporter, that is transactivated by the retinoic acid receptor (RAR)/RXRα, was also decreased. Previous studies have shown that arsenic in the concentration range of 2-5 µM affects CYP3A mRNA. When rifampicin-treated primary human hepatocyte cultures were exposed to arsenite concentrations as low as 50 nM, CYP3A mRNA was decreased. Treatment of primary human hepatocytes with the proteasome inhibitor MG132 increased RXRα suggesting the involvement of the proteasome pathway in regulation of RXRα. Finally, arsenic induces a pro-inflammatory response in liver. Surprisingly, we show that in hepatocytes arsenite decreases expression of two inflammatory mediators, TNF and VEGF, an effect that is not predicted from suppression of RXRα activity.

Inherent and benzo[a]pyrene-induced differential aryl hydrocarbon receptor signaling greatly affects life span, atherosclerosis, cardiac gene expression, and body and heart growth in mice.

Little is known of the environmental factors that initiate and promote disease. The aryl hydrocarbon receptor (AHR) is a key regulator of xenobiotic metabolism and plays a major role in gene/environment interactions. The AHR has also been demonstrated to carry out critical functions in development and disease. A qualitative investigation into the contribution by the AHR when stimulated to different levels of activity was undertaken to determine whether AHR-regulated gene/environment interactions are an underlying cause of cardiovascular disease. We used two congenic mouse models differing at the Ahr gene, which encodes AHRs with a 10-fold difference in signaling potencies. Benzo[a]pyrene (BaP), a pervasive environmental toxicant, atherogen, and potent agonist for the AHR, was used as the environmental agent for AHR activation. We tested the hypothesis that activation of the AHR of different signaling potencies by BaP would have differential effects on the physiology and pathology of the mouse cardiovascular system. We found that differential AHR signaling from an exposure to BaP caused lethality in mice with the low-affinity AHR, altered the growth rates of the body and several organs, induced atherosclerosis to a greater extent in mice with the high-affinity AHR, and had a huge impact on gene expression of the aorta. Our studies also demonstrated an endogenous role for AHR signaling in regulating heart size. We report a gene/environment interaction linking differential AHR signaling in the mouse to altered aorta gene expression profiles, changes in body and organ growth rates, and atherosclerosis.

Differential regulation of polysome mRNA levels in mouse Hepa-1C1C7 cells exposed to dioxin.

The environmental agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD or dioxin) causes a multitude of human illnesses. In order to more fully understand the underlying biology of TCDD toxicity, we tested the hypothesis that new candidate genes could be identified using polysome RNA from TCDD-treated mouse Hepa-1c1c7 cells. We found that (i) differentially expressed whole cell and cytoplasm RNA levels are both poor predictors of polysome RNA levels; (ii) for a majority of RNAs, differential RNA levels are regulated independently in the nucleus, cytoplasm, and polysomes; (iii) for the remaining polysome RNAs, levels are regulated via several different mechanisms, including a "tagging" of mRNAs in the nucleus for immediate polysome entry; and (iv) most importantly, a gene list derived from differentially expressed polysome RNA generated new genes and cell pathways potentially related to TCDD biology.

miRNAs in newt lens regeneration: specific control of proliferation and evidence for miRNA networking.

BACKGROUND: Lens regeneration in adult newts occurs via transdifferentiation of the pigment epithelial cells (PECs) of the dorsal iris. The same source of cells from the ventral iris is not able to undergo this process. In an attempt to understand this restriction we have studied in the past expression patterns of miRNAs. Among several miRNAs we have found that mir-148 shows an up-regulation in the ventral iris, while members of the let-7 family showed down-regulation in dorsal iris during dedifferentiation. METHODOLOGY/PRINCIPAL FINDINGS: We have performed gain- and loss-of-function experiments of mir-148 and let-7b in an attempt to delineate their function. We find that up-regulation of mir-148 caused significant decrease in the proliferation rates of ventral PECs only, while up-regulation of let-7b affected proliferation of both dorsal and ventral PECs. Neither miRNA was able to affect lens morphogenesis or induction. To further understand how this effect of miRNA up-regulation is mediated we examined global expression of miRNAs after up-regulation of mir148 and let-7b. Interestingly, we identified a novel level of mirRNA regulation, which might indicate that miRNAs are regulated as a network. CONCLUSION/SIGNIFICANCE: The major conclusion is that different miRNAs can control proliferation in the dorsal or ventral iris possibly by a different mechanism. Of interest is that down-regulation of the let-7 family members has also been documented in other systems undergoing reprogramming, such as in stem cells or oocytes. This might indicate that reprogramming during newt regeneration shares common molecular signatures with reprogramming in stem or germ cells. On the other hand that miRNAs can regulate the levels of other miRNAs is a novel level of regulation, which might provide new insights on their function.

Microarray analysis of cytoplasmic versus whole cell RNA reveals a considerable number of missed and false positive mRNAs.

With no known exceptions, every published microarray study to determine differential mRNA levels in eukaryotes used RNA extracted from whole cells. It is assumed that the use of whole cell RNA in microarray gene expression analysis provides a legitimate profile of steady-state mRNA. Standard labeling methods and the prevailing dogma that mRNA resides almost exclusively in the cytoplasm has led to the long-standing belief that the nuclear RNA contribution is negligible. We report that unadulterated cytoplasmic RNA uncovers differentially expressed mRNAs that otherwise would not have been detected when using whole cell RNA and that the inclusion of nuclear RNA has a large impact on whole cell gene expression microarray results by distorting the mRNA profile to the extent that a substantial number of false positives are generated. We conclude that to produce a valid profile of the steady-state mRNA population, the nuclear component must be excluded, and to arrive at a more realistic view of a cell's gene expression profile, the nuclear and cytoplasmic RNA fractions should be analyzed separately.

Effect of proteasome inhibition on toxicity and CYP3A23 induction in cultured rat hepatocytes: comparison with arsenite.

Previous work in our laboratory has shown that acute exposure of primary rat hepatocyte cultures to non-toxic concentrations of arsenite causes major decreases in the DEX-mediated induction of CYP3A23 protein, with minor decreases in CYP3A23 mRNA. To elucidate the mechanism for these effects of arsenite, the effects of arsenite and proteasome inhibition, separately and in combination, on induction of CYP3A23 protein were compared. The proteasome inhibitor, MG132, inhibited proteasome activity, but also decreased CYP3A23 mRNA and protein. Lactacystin, another proteasome inhibitor, decreased CYP3A23 protein without affecting CYP3A23 mRNA at a concentration that effectively inhibited proteasome activity. This result, suggesting that the action of lactacystin is similar to arsenite and was post-transcriptional, was confirmed by the finding that lactacystin decreased association of DEX-induced CYP3A23 mRNA with polyribosomes. Both MG132 and lactacystin inhibited total protein synthesis, but did not affect MTT reduction. Arsenite had no effect on ubiquitination of proteins, nor did arsenite significantly affect proteasomal activity. These results suggest that arsenite and lactacystin act by similar mechanisms to inhibit translation of CYP3A23.

Effect of insulin and glucagon on accumulation of uroporphyrin and coproporphyrin from 5-aminolevulinate in hepatocyte cultures.

Primary cultures of chick embryo hepatocytes have been used to study the mechanisms by which various drugs and other chemicals cause accumulation of porphyrin intermediates of the heme pathway. When these cultures are incubated with the heme precursor, 5-aminolevulinic acid (ALA), there is a major accumulation of protoporphyrin. However, in the presence of ALA, addition of insulin caused a striking increase in accumulation of uroporphyrin I and coproporphyrin III, whereas addition of glucagon mainly caused an increase in uroporphyrin I. Treatment with both insulin and glucagon resulted in additive increases in uroporphyrin, but not coproporphyrin. Antioxidants abolished the uroporphyrin I accumulation and increased coproporphyrin III. Insulin caused an increase in uptake of ALA and an increase in porphobilinogen accumulation, suggesting that the accumulation of uroporphyrin I is due to increased flux through the heme pathway. Apparently, this increased flux could particularly affect the utilization of the intermediate hydroxymethylbilane, which would result in accumulation of uroporphyrin I.

Mechanism of arsenite-mediated decreases in CYP3A23 in rat hepatocytes.

In primary cultures of rat hepatocytes, exposure to arsenite causes a major decrease in dexamethasone (DEX)-mediated induction of CYP3A23 hemoprotein, with a minor decrease in CYP3A23 mRNA. Here we show that addition of heme did not prevent the arsenite-mediated decreases in CYP3A23 protein, and arsenite did not decrease intracellular glutathione levels, indicating that heme and glutathione were not limiting for formation of holoCYP3A23. We also investigated whether arsenite decreases CYP3A23 protein by increasing CYP3A23 degradation by the calpain pathway. The calpain inhibitor, calpeptin, caused greater than a 90% inhibition of calpain-mediated proteolysis, but had no effect on DEX-mediated induction of CYP3A23 protein following 24h treatments. However, calpeptin enhanced the effect of arsenite to decrease induction of CYP3A23 protein. In addition, in short-term studies, calpeptin appeared to be a suicidal inhibitor of CYP3A-catalyzed enzyme activity. Our findings suggest that CYP3A23 protein is not degraded by calpain-mediated proteolysis, even in the presence of arsenite.

Arsenite decreases CYP3A23 induction in cultured rat hepatocytes by transcriptional and translational mechanisms.

Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (CYP) levels by arsenic. In rat and human hepatocyte cultures, arsenic, in the form of arsenite, decreases the induction of several CYPs. The present study investigated whether arsenite utilizes transcriptional or post-transcriptional mechanisms to decrease CYP3A23 in primary cultures of rat hepatocytes. In these cultures, a 6-h treatment with 5 microM arsenite abolished dexamethasone (DEX)-mediated induction of CYP3A23 protein and activity, but did not inhibit general protein synthesis. However, arsenite treatment only reduced DEX-induced levels of CYP3A23 mRNA by 30%. The effects of arsenite on CYP3A23 transcription were examined using a luciferase reporter construct containing 1.4 kb of the CYP3A23 promoter. Arsenite caused a 30% decrease in DEX-induced luciferase expression of this reporter. Since arsenite abolished induction of CYP3A23 protein, but caused only a small decrease in CYP3A23 mRNA, the effects of arsenite on translation of CYP3A23 mRNA were investigated. Polysomal distribution analysis showed that arsenite decreased translation by decreasing the DEX-mediated increase in CYP3A23 mRNA association with polyribosomes. Arsenite did not decrease intracellular glutathione or increase lipid peroxidation, suggesting that the effect of arsenite on CYP3A23 does not involve oxidative stress. Overall, the results suggest that low-level arsenite decreases both transcription and translation of CYP3A23 in primary rat hepatocyte cultures.

Arsenite decreases CYP3A4 and RXRalpha in primary human hepatocytes.

Arsenic is a naturally occurring, worldwide contaminant implicated in numerous pathological conditions in humans, including cancer and several forms of liver disease. One of the contributing factors to these disorders may be the alteration of cytochrome P450 (P450) levels by arsenic. P450s are involved in the oxidative metabolism and elimination of numerous toxic chemicals. CYP3A4, a major P450 in humans, is involved in the metabolism of half of all currently used drugs. Acute exposure to arsenite decreases the induction of CYP1A1/2 proteins and activities in cultured human hepatocytes, as well as CYP3A23 in cultured rat hepatocytes. Here, in primary cultures of human hepatocytes, we assessed the effects of acute arsenite exposure on CYP3A4 and several transcription factors involved in CYP3A4 expression. The concentrations of arsenite used in these studies were nontoxic to the hepatocytes and failed to elicit an oxidative response. Treatment with arsenite in the presence of CYP3A4 inducers, rifampicin (Rif) or phenobarbital, caused major decreases in CYP3A4 mRNA, protein, and activity. In addition, the levels of CYP3A4 in untreated cells were decreased following arsenite treatment. Transcription of the CYP3A4 gene is primarily regulated by heterodimers of the retinoid X receptor alpha (RXRalpha) and the pregnane X receptor (PXR). We found that arsenite failed to affect expression of PXR or the transcription factor Sp1, yet caused a significant decrease in PXR responsiveness to Rif. Arsenite caused a large decrease in nuclear RXRalpha protein and, to a lesser extent, RXRalpha mRNA. These results suggest that arsenite inhibits both untreated and induced CYP3A4 transcription in primary human hepatocytes by decreasing the activity of PXR, as well as expression of the nuclear receptor RXRalpha.

Genetic factors influence ethanol-induced uroporphyria in Hfe(-/-) mice.

Two major risk factors for porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). We recently described an animal model for alcohol-induced uroporphyria, using Hfe(-/-) mice. In the present study we show that this effect is dependent on genetic background and ethanol dose. In the 129S6/SvEvTac (129) strain, treatment with 15% ethanol in the drinking water for 6.5 months produced an accumulation of hepatic uroporphyrin (URO) 4-fold higher than that observed with 10% ethanol, a 90% decrease in uroporphyrinogen decarboxylase activity (UROD), and further increased the activities of hepatic 5-aminolevulinate synthase (ALAS) and CYP1A2. Hepatic nonheme iron (NHFe) and hepatocyte iron staining were not further increased by 15% compared to 10% ethanol. Treatment of C57BL/6 Hfe(-/-) mice with 15% ethanol for 6.5 months did not increase hepatic URO. Although NHFe was increased by ethanol, the resulting level was only half that of ethanol-treated 129 Hfe(-/-) mice. ALAS induction was similar in both Hfe(-/-) strains. In wild-type 129 mice treated with ethanol for 6 to 7 months, administration of iron dextran increased hepatic URO accumulation and decreased UROD activity. In conclusion, this study demonstrates a strong effect of genetic background on ethanol-induced uroporphyria, which is probably due to a greater effect of ethanol on iron metabolism in the susceptible strain.

Uroporphyrin accumulation in hepatoma cells expressing human or mouse CYP1A2: relation to the role of CYP1A2 in human porphyria cutanea tarda.

In experimental animals, CYP1A2 is absolutely required for the development of uroporphyria induced by treatment with polyhalogenated aromatic compounds or other compounds. Although the role of this CYP in clinical uroporphyria, porphyria cutanea tarda (PCT), is not clear, Cyp1a2(-/-) mice are resistant to the development of uroporphyria. Here, we compared the abilities of human and mouse CYP1A2 expressed in mouse hepatoma Hepa-1 cells to: (i) catalyze CYP1A2-dependent methoxyresorufin demethylase (MROD), and (ii) support uroporphyrin (URO) accumulation. Both CYP1A2 orthologs were expressed at similar levels as indicated by immunodetectable CYP1A2 proteins and MROD activities. URO accumulation was increased in cultures expressing either ortholog when supplemented with 5-aminolevulinic acid, the porphyrin precursor. Cells expressing mouse CYP1A2 produced more URO than cells expressing human CYP1A2. The results indicate that human CYP1A2 can support URO accumulation in hepatoma cells and thus may play a role in human PCT.

Uroporphyria caused by ethanol in Hfe(-/-) mice as a model for porphyria cutanea tarda.

Two major risk factors for the development of porphyria cutanea tarda (PCT) are alcohol consumption and homozygosity for the C282Y mutation in the hereditary hemochromatosis gene (HFE). To develop an animal model, Hfe knockout mice were treated continuously with 10% ethanol in drinking water. By 4 months, uroporphyrin (URO) was detected in the urine. At 6 to 7 months, hepatic URO was increased and hepatic uroporphyrinogen decarboxylase (UROD) activity was decreased. Untreated Hfe(-/-) mice or wild-type mice treated with or without ethanol did not show any of these biochemical changes. Treatment with ethanol increased hepatic nonheme iron and hepatic 5-aminolevulinate synthase activity in Hfe(-/-) but not wild-type mice. The increases in nonheme iron in Hfe(-/-) mice were associated with diffuse increases in iron staining of parenchymal cells but without evidence of significant liver injury. In conclusion, the results of this study suggest that the uroporphyrinogenic effect of ethanol is mediated by its effects on hepatic iron metabolism. Ethanol-treated Hfe(-/-) mice seem to be an excellent model for studies of alcohol-mediated PCT.