The dysfunction of hepatic heme synthesis by 2,3,7,8-tetrachlordibenzo- p-dioxin (TCDD) in mice, enhanced by iron, leads to accumulation of uroporphyrins I and III (uroporphyria) and resembles the human disorder porphyria cutanea tarda (PCT) precipitated by alcohol and estrogenic drugs. Although consequences of TCDD are considered entirely dependent on the aryl hydrocarbon receptor (AHR), this is not proven for uroporphyria. Administration of TCDD (75 microg/kg) caused uroporphyria in susceptible C57BL/6J mice with high-affinity AHR after 5 weeks (>600-fold increase in hepatic uroporphyrins). Transcriptomics showed significant modified gene expressions for intermediary, heme, and iron metabolism as well as for oxidative stress and cell injury. Resistant low-affinity AHR DBA/2 mice (no increase in porphyrins) showed far fewer changes. At this dose of TCDD, persistent up-regulation of some traditional AH battery genes occurred in both strains. Essentiality of AHR was demonstrated with C57BL/6 Ahr knockout mice. Elevation of hepatic uroporphyrins was 964-fold in Ahr (+/+) mice, lower in Ahr (+/-) (60-fold), but undetectable with Ahr (-/-) . Consistent with an oxidative mechanism, iron overload enhanced porphyria as well as general liver injury in Ahr (+/+) and Ahr (+/-) mice but had no interactive effect in Ahr (-/-) . In contrast, when iron-treated mice received, instead of TCDD, the heme precursor 5-aminolevulinic acid (ALA), causing uroporphyia in Ahr (+/+) mice (242-fold rise in uroporphyrins), elevation of uroporphyrins I and III (42-fold) also occurred in Ahr (-/-) mice and was seemingly associated with AHR-independent expression of Cyp1a2. The findings prove that AHR is a key factor in porphyria induced in mice by TCDD. However, in other models of human PCT, participation of AHR may not be an essential requirement.

5-Aminolevulinic acid synthase 1 (ALAS1) is the key enzyme in the homeostasis of nonerythroid heme and of fundamental importance in respiration, the metabolism of drugs, chemicals and steroids and cell signalling. The regulation of ALAS1 in response to stimuli occurs at transcriptional, translational and post-translational levels which could depend on inter-individual variation in basal expression. A genetic difference in hepatic ALAS1 mRNA levels between C57BL/6J and DBA/2 mice was detected by microarray and was >5-fold in whole liver or hepatocytes when estimated by qRT-PCR. Analysis of the ALAS1 promoter showed a 210 nt insert in the DBA/2 containing a B2 SINE retrotransposon causing a marked repression of expression by intracellular reporter systems. Deletions across the B2 SINE demonstrated that the full sequence was required for transcriptional inhibition. The findings show that a B2 SINE can contribute to the regulation of ALAS1 and SINEs in 5'-UTR regions contribute to inter-individual differences in gene expression.

Polymorphisms of genes linked to iron metabolism may account for individual variability in hemochromatosis and iron status connected with liver and cardiovascular diseases, cancers, toxicity, and infection. Mouse strains exhibit marked differences in levels of non‐heme iron, with C57BL/6J and SWR showing low and high levels, respectively. The genetic basis for this variability was examined using quantitative trait loci (QTL) analysis together with expression profiling and chromosomal positions of known iron‐related genes. Non‐heme iron levels in liver and spleen of C57BL/6J × SWR F2 mice were poorly correlated, indicating independent regulation. Highly significant (P < .01) polymorphic loci were found on chromosomes 2 and 16 for liver and on chromosomes 8 and 9 for spleen. With sex as a covariate, additional significant or suggestive (P < 0.1) QTL were detected on chromosomes 7, 8, 11, and 19 for liver and on chromosome 2 for spleen. A gene array showed no clear association between most loci and differential iron‐related gene expression. The gene for transferrin and a transferrin‐like gene map close to the QTL on chromosome 9. Transferrin saturation was significantly lower in C57BL/6J mice than in SWR mice, but there was no significant difference in the serum level of transferrin, hepatic expression, or functional change in cDNA sequence. β2‐Microglobulin, which, unlike other loci, was associated with C57BL/6J alleles, is a candidate for the chromosome 2 QTL for higher iron. In conclusion, the findings show the location of polymorphic genes that determine basal iron status in wild‐type mice. Human equivalents may be pertinent in predisposition to hepatic and other disorders. (HEPATOLOGY 2006;44:174–185.)

We have previously used genome-wide transcript profiling to investigate the relationships between changes in gene expression and physiological alterations during the response of the immature mouse uterus to estrogens. Here we describe the identification of a functionally inter-related group of estrogen-responsive genes associated with iron homeostasis, including the iron-binding protein lactotransferrin, the ferroxidase ceruloplasmin, the iron delivery protein lipocalin 2 and the iron-exporter ferroportin. Quantitative real-time PCR revealed that the expression of these genes increases with time during the uterotrophic response, reaching maximal levels in the post-proliferative phase (between 48 and 72 h). In contrast, the heme biosynthesis genes aminolevulinic acid synthase 1 and 2 were maximally induced by estrogen at 2 and 4 h, respectively, prior to increased cell proliferation. Together, these data reveal that estrogen induces the temporally coordinated expression of iron homeostasis genes in the mouse uterus, and suggest an important role for iron metabolism during sex steroid hormone-induced uterine cell growth and differentiation.

BALB/c Fech(m1Pas) mice have a mutated ferrochelatase gene resulting in protoporphyria that models the hepatic injury occurring sporadically in human erythropoietic protoporphyria. We used this mouse model to study the development of the injury and to compare the dysfunction of heme synthesis with hepatic gene expression of liver metabolism, oxidative stress, and cellular injury/inflammation. From an early age expression of total cytochrome P450 and many of its isoforms was significantly lower than in wild-type mice. However, despite massive accumulation of protoporphyrin in the liver, expression of the main genes controlling heme synthesis and catabolism (Alas1 and Hmox1, respectively) were only modestly affected even in the presence of the cytochrome P450-inducing CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. In contrast, in BALB/c mice exhibiting griseofulvin-induced hepatic protoporphyria with induction and destruction of cytochrome P450, both Alas1 and Hmox1 genes were markedly up-regulated. Other expression profiles in BALB/c Fech(m1Pas) mice identified roles for oxidative mechanisms in liver injury while modulated gene expression of hepatocyte transport proteins and cholesterol and bile acid synthesis illustrated the development of cholestasis. Subsequent inflammation and cirrhosis were also shown by the up-regulation of cytokine, cell cycling, and procollagen genes. Thus, gene expression profiles studied in Fech(m1Pas) mice may provide candidates for human polymorphisms that explain the sporadic hepatic consequences of erythropoietic protoporphyria.

Aryl hydrocarbon receptor ligands, such as polychlorinated biphenyls (PCBs), cause inhibition of the heme biosynthesis enzyme, uroporphyrinogen decarboxylase; this leads to uroporphyria and hepatic tumors, which are markedly enhanced by iron overload in C57BL/10 and C57BL/6 strains of mice. Cyp1a2(-/-) knockout mice were used to compare the effects of CYP1A2 expression on uroporphyria and liver carcinogenesis. PCBs in the diet (100ppm) of Cyp1a2(+/+) wild-type mice caused hepatic uroporphyria, which was strongly increased by iron-dextran (800mg Fe/kg). In contrast, uroporphyria was not detected in Cyp1a2(-/-) knockout mice, although expression of CYP1A1 and CYP2B10 was greatly induced. After 57 weeks on this diet, hepatic preneoplastic foci and tumors were seen in the Cyp1a2(+/+) mice; numbers and severity were enhanced by iron. No foci or tumors were detected in Cyp1a2(-/-) mice, although evidence for other forms of liver injury was observed. Our findings suggest a link not only between CYP1A2, iron metabolism, and the induction of uroporphyria by PCBs, but also with subsequent hepatocarcinogenesis.

Synchronization of circadian oscillators with the outside world is achieved by the acute effects of light on the levels of one or more clock components. In mammals the PAS transcription factors Clock, NPAS2, and BMAL1 regulate gene expression as a function of the day‐night cycle. Both PAS domains of NPAS2 were found to bind heme as a prosthetic group, form a gas‐regulated sensor, and exert heme‐status control of DNA binding in vitro. In a microarray analysis comparing overall changes in brain transcript levels between mice subjected to light pulses during the dark phase with animals maintained in darkness, we traced consistent changes in more than 200 different transcripts. Of these, 20 are associated with heme and iron biosynthesis and catabolism. A model for the pathway of induction of heme and iron homeostasis‐related transcripts resulting from light pulses suggests that light signals (as stressors) induce transcription of heme oxygenase 2 (Hmox2) and cytochrome P450 oxidoreductase (Por), which may serve as a primary line of cellular defense. HMOX2 degrades heme from proteins such as hemoglobin. This degradation generates CO, a signal molecule, and may also change the redox state of the cell by reducing the NADPH/NADP ratio. This could lead to up‐regulation of globin gene transcription, thereby releasing iron that in turn controls production of ferritins, and further up‐regulating aminolevulinate synthase 2 (Alas2).

Differential gene expression in two established initiation and promotion skin carcinogenesis models during promotion and tumor formation was determined by microarray technology with the purpose of distinguishing the genes more associated with neoplastic transformation from those linked with proliferation and differentiation. The first model utilized dimethylbenz[a]anthracene initiation and 12-O-tetradecanoylphorbol 13-acetate (TPA) promotion in the FVB/N mouse, and the second TPA promotion of the Tg.Ac mouse, which is endogenously initiated by virtue of an activated Ha-ras transgene. Comparison of gene expression profiles across the two models identified genes whose altered expression was associated with papilloma formation rather than TPA-induced proliferation and differentiation. DMBA suppressed TPA-induced differentiation which allowed identification of those genes associated more specifically with differentiation rather than proliferation. EASE (Expression Analysis Systemic Explorer) indicated a correlation between muscle-associated genes and skin differentiation, whereas genes involved with protein biosynthesis were strongly correlated with proliferation. For verification the altered expression of selected genes were confirmed by RT-PCR; Carbonic anhydrase 2, Thioredoxin 1 and Glutathione S-transferase omega 1 associated with papilloma formation and Enolase 3, Cystatin beta and Filaggrin associated with TPA-induced proliferation and differentiation. In situ analysis located the papillomas Glutathione S-transferase omega 1 expression to the proliferating areas of the papillomas. Thus we have identified profiles of differential gene expression associated with the tumorigenesis and promotion stages for skin carcinogenesis in the mouse.

The molecular mechanisms of toxic agents are rarely likely to be the result of change in the expression of a single gene. Even apparently simple actions of chemicals are probably the consequence of genetically variable multigene expression. One strategy for attacking this problem is to take advantage of the genetic variation of response in mice to search for susceptibility genes using genetic linkage analysis in combination with comparisons of gene expression in the parent strains by cDNA microarray technology. Genetic variation in mice and toxicogenomics were used to explore mechanisms of gene interaction leading to cell malfunction and injury in the liver caused by dioxin. This demonstrated susceptibility loci, other than the Ahr gene, pertinent to the development of porphyria (a disruption of heme synthesis) and liver injury. cDNA arrays of 4000 IMAGE clones pertinent to toxicology were used to compare candidate multiple gene expression in strains relative to their initial hepatic response e.g. induction of drug metabolism enzymes, and to their subsequent development of porphyria and liver injury. Phenotypic response was compared with gene expression by metabolic system including groups of genes for heme and iron metabolism, the AH battery and oxidative stress.

One of the major complications of cancer chemotherapy is the development of a multidrug resistance (MDR) phenotype. Genes known to be major contributors to this phenotype include the ABC transporter family, DNA repair class genes and phase II conjugators such as the GST family. However, though there may be predominant contribution to the MDR phenotype from one gene or family the overall phenotype probably results from the contribution of a cohort of genes, both of downstream effecter and upstream regulatory classes. Additionally, other genes differentially expressed in the MDR verses drug sensitive states may result in other phenotypic traits that may also complicate treatment, for example an increased propensity towards invasiveness. We have applied microarray analysis to breast carcinoma cells with intrinsic and acquired doxorubicin resistant phenotypes and compared the gene expression profiles obtained with each other, and with those gathered from various other drug sensitive breast carcinoma cell lines (Turton et al. 2001). The analysis has been applied at both the mRNA and genomic levels. Some expected changes in gene expression (mRNA analysis) and amplification (genomic DNA analysis) such as that for the multidrug resistance 1 (ABCB1) gene were detected. However, some unexpected associations of differential gene expression were found, some associated with known phenotypic traits, and some that may offer alternative targets for chemotherapy. The similarity of gene expression profiles between the cells with intrinsic and acquired doxorubicin resistance indicated that a tumor with an acquired doxorubicin resistant phenotype arises as a result of drug driven selection of cells with an intrinsic resistant phenotype from the majority drug sensitive population.

Erythropoietic protoporphyria patients can develop cholestasis, severe hepatic damage, fibrosis, and cirrhosis. We modeled this hepatic pathology in C57BL/6J and BALB/c mice using griseofulvin and analyzed 3,127 genes for alteration of expression in the liver before and during the onset of protoporphyria, cholestasis, inflammation, and hepatic fibrosis. The two mouse strains developed different levels of pathologic damage in response to the griseofulvin. Characteristic gene expression profiles could be associated with griseofulvin-induced gene expression, disruption of lipid metabolism, and the pathologic states of inflammation, early fibrosis, and cholestasis. Additionally, some genes individually indicated an alteration of homeostasis. or pathologic state; for example, fibroblast proliferation was potentially indicated by increased calcyclin (SA100a6) expression. Changes in cytochrome P450 (Cyp) gene expression were particularly pronounced, with increased expression of the Cyp2a, Cyp2b, and Cyp3a families. Decreased Cyp4a10 and Cyp4a14 expression was observed that could be associated with early pathologic change. A potential decrease in bile acid and steroid biosynthesis was indicated by the decreased expression of Cyp7b1 and Hsd3b4, respectively. DNA damage was indicated by induction of GADD45. This study illustrates how transcriptional programs can be associated with different stimuli in the same experiment. The time course of change in the gene expression profile compared with changes in pathology and clinical chemistry shows the potential of this approach for modeling causative, predictive, and adaptive changes in gene expression during pathologic change.

Ecteinascidin-743 (ET-743) is a novel marine-derived anticancer drug with clinical activity in soft tissue sarcoma and ovarian cancer. Reversible transaminitis and subclinical cholangitis have frequently been described in patients who receive ET-743. To facilitate understanding of this adverse effect and help design suitable therapeutic rescue strategies, we characterized the hepatic effects of ET-743 in rats. Female rats received ET-743 (single dose, 40 microg/kg) i.v., and liver changes were assessed from 6 h up to 3 months after dosing by histopathology, immunohistochemistry, electron microscopy, hepatic and plasma biochemistry, and DNA microarray analysis. At 24 h posttreatment and beyond, livers displayed degeneration and patchy focal necrosis of bile duct epithelial cells associated with mild inflammation followed by fibrosis. Sporadic and focal zones of hepatic necrosis and hemorrhage were observed from day 2 onward, although the majority of hepatocytes appeared normal as judged by electron microscopy. Pathological alterations persisted up to 3 months after dosing. Plasma levels of total bilirubin were elevated up to 7-fold over those in untreated rats from day 2 onward and returned to control values by day 24. Activities of alkaline phosphatase and aspartate aminotransferase in plasma were elevated for 2 and 3 months, respectively. Activities of the hepatic microsomal drug-metabolizing enzymes cytochrome P-450 A1/2, CYP2E1, and CYP3A2 were decreased. DNA microarray analysis of livers from ET-743-treated animals showed a dramatic increase in the expression of ATP binding cassette transport genes Abcb1a and Abcb1b, which impart resistance to anticancer drugs, and of Cdc2a and Ccnd1, the rodent homologues of human cell cycle genes CDC2 and cyclin D1, respectively. The cell cycle gene expression changes mirrored ET-743-induced increases in liver weight and Ki-67 labeling of liver nuclei. The results suggest that the toxicity exerted by ET-743 in the rat liver is a consequence of biliary rather than hepatocellular damage and that it is accompanied by a wave of mitogenic activity, which may be driven by the transcriptional increase in Cdc2a expression.

Administration of tamoxifen (TAM) (20 mg/kg per day p.o.) for 6 weeks to female lambda/lacI transgenic rats caused a 4-fold increase in mutation frequency (MF) at the lacI gene locus in the livers of dosed animals compared with controls. After cessation of dosing, the MF showed a further increase with time at 2, 12 and 24 weeks, respectively. Phenobarbital promotion of similarly treated animals resulted in no increase in mutation frequency compared with TAM alone. Treatment with phenobarbital or TAM+phenobarbital resulted in time-dependent increases in liver weight compared with the corresponding controls. There was an increase in cell proliferation in the phenobarbital and TAM+phenobarbital groups, and at 24 weeks in the TAM dosed animals compared with controls. There was also a progressive increase in the number of GST-P expressing foci in the livers of TAM and TAM + phenobarbital rats compared with controls. The induction of cell proliferation and GSTP foci in the rat liver by phenobarbital is consistent with its ability to promote tamoxifen-initiated liver tumours in the rat. If the lacI gene is regarded as being representative of the rat genome in general (albeit that the gene is bacterial) the above observations suggest that promotion by tamoxifen confers selective advantage on mutated genes at loci that contribute to the tumour phenotype and that promotion of rat liver tumours by tamoxifen is not dependent simply upon the enhancement of cellular proliferation.

It is now generally accepted that activation of tamoxifen occurs as a result of metabolism to alpha-hydroxytamoxifen. In this study, alpha-hydroxytamoxifen was given to female Wistar/Han rats (0.103 or 0.0103 mmol/kg, intraperitoneally, daily for 5 days). This resulted in liver DNA damage, determined by (32)P-post-labelling, of 3333 +/- 795 or 343 +/- 68 adducts/10(8) nucleotides, respectively (mean +/- SD, n = 4). Following HPLC separation, the retention times of the major alpha-hydroxytamoxifen DNA adducts were similar to those seen following the administration of tamoxifen. However, after rats were treated with alpha-hydroxytamoxifen (0.103 mmol/kg) for 5 days and the animals kept for up to 13 months, no liver tumours developed (0/7 rats), even with phenobarbital promotion (0/5 rats). GST-P foci were detected in the liver, but only after 13 months was their number or area significantly increased over the corresponding controls. When alpha-hydroxytamoxifen was given to female lambda/lacI transgenic rats (0.103 mmol/kg orally for 10 days) and the animals killed 46 days later, there was an approximate 1.8-fold increase in mutation frequency but no significant increase in G:C to T:A transversions as described after tamoxifen treatment. It is concluded that DNA damage alone, resulting from the short-term administration of alpha-hydroxytamoxifen, is not sufficient to initiate liver tumours even with phenobarbital promotion. As with tamoxifen, long-term exposure may be required to allow promotion and progression of transformed cells.

The anti-oestrogenic drug tamoxifen has been under investigation as a breast cancer chemopreventive agent for at least a decade. However, its use for this purpose is still debatable since it is able to induce liver tumours in rats via a mechanism involving metabolic activation to a DNA adduct-forming electrophilic intermediate. The metabolic activation and adduct-forming properties of tamoxifen are now well characterized but less is known about its ability to induce hepatic cell proliferation, which is also essential for the carcinogenic process. The effects of tamoxifen on liver weight and cell proliferation were compared in female Fischer 344 (F344), Wistar and Lewis rats given the drug in the diet for up to 26 weeks. The onset and duration of hepatic cell proliferation varied between the strains of rat. In Wistar and Lewis but not F344 rats there was a marked increase in hepatocellular proliferation during the first 4 weeks of tamoxifen administration. In the Wistar strain this was associated with an increase in DNA adduct levels; no such increase was observed in the F344 strain. The onset of the proliferative response was delayed until the 13 week time point in the F344 strain. By the 13 and 26 week time points, cell proliferation in tamoxifen-treated Wistar and Lewis rat liver had returned to normal, but the amount of apoptotic activity in these livers was elevated. This suggests that excess cells generated during the proliferative phase of tamoxifen treatment were being eliminated by apoptosis. In the F344 strain, however, increased proliferative activity was associated with relatively low apoptotic activity at the 26 week time point, suggesting that the delayed proliferative response had yet to be balanced by apoptotic deletion. This is consistent with the fact that tamoxifen-induced hepatocellular tumours develop very late, towards the end of the lifespan, in this strain. The cell proliferative activity of tamoxifen in the Wistar rat liver was compared with that of a non-mutagenic analogue, toremifene. Tamoxifen induced increased cell cycle activity in the livers of rats following gavage dosing at all sampling times (1–12 weeks), whereas toremifene had no effect on the incidence of cycling in hepatic cells, demonstrating that the hepatic cell proliferation is not a general response to anti-oestrogen treatment. These observations suggest that the rate of promotion of liver tumours by tamoxifen is a function of the rate, time of onset and duration of increased cell replication. The susceptibility of rat strains to the hepatocarcinogenic effects of tamoxifen appears to depend upon the balance between initiation via DNA adduct formation, promotion via increased cell proliferation and cell deletion via apoptosis. Our findings suggest that an early proliferative response to tamoxifen is important in this process.

The multidrug resistance (MDR) phenotype is a major cause of cancer treatment failure. Here the expressions of 4224 genes were analysed for association with intrinsic or acquired doxorubicin (DOX) resistance. A cluster of overexpressed genes related to DOX resistance was observed. Included in this cluster was ABCB1 the P-glycoprotein transporter protein gene and MMP1 (Matrix Metalloproteinase 1), indicative of the invasive nature of resistant cells, and the oxytocin receptor (OXTR), a potential new therapeutic target. Overexpression of genes associated with xenobiotic transformation, cell transformation, cell signalling and lymphocyte activation was also associated with DOX resistance as was estrogen receptor negativity. In all carcinoma cells, compared with HBL100 a putatively normal breast epithelial cell line, a cluster of overexpressed genes was identified which included several keratins, in particular keratins 8 and 18 which are regulated through the ras signalling pathway. Analysis of genomic amplifications and deletions revealed specific genetic alterations common to both intrinsic and acquired DOX resistance including ABCB1, PGY3 (ABCB4) and BAK. The findings shown here indicate new possibilities for the diagnosis of DOX resistance using gene expression, and potential novel therapeutic targets for pharmacological intervention.

The synergistic interaction of iron overload, AHR: genotype and exposure to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1254) in mice leads to hepatic porphyria, oxidative DNA damage and cancer. In humans, hepatocellular cancer is associated with iron overload and hepatic porphyria. Neither the mechanism of hepatic carcinogenesis induced by PCBs in rodents nor hepatocellular cancer induced by iron and porphyria in humans are understood. To test the hypothesis that chronic interaction of iron and PCBs may induce mutagenesis in liver DNA, lambda /lacI transgenic C57BL/6 mice were given iron dextran (600 mg iron/kg) and then administered Aroclor 1254 in the diet (0.01%) for 7 weeks. Hepatic iron, CYP1A activity and CYP1A1/1A2 protein were elevated >20-fold as a result of iron or Aroclor treatments, respectively, but porphyria with associated histological changes only developed in the combined iron/Aroclor treatment group. lambda/lacI shuttle vectors were isolated from liver genomic DNA and the mutational frequency (MF) in the lacI gene determined. Both iron and Aroclor treatments alone caused significant small increases in MF (1.5- and 1.4-fold, respectively), however, the MF following the combined iron and Aroclor treatment (1. 6-fold) was not greater than the additive effects. In contrast, the MF was significantly elevated (4.7-fold) in liver DNA of mice 2 weeks following five daily doses of N-nitrosodimethylamine (4 mg/kg). These studies demonstrate that neither PCBs nor iron overload caused marked point mutations even in a combination regime that leads to oxidative damage and cancer. There was also no strong evidence either that porphyrins or chronic CYP1A1 expression induced by the PCBs after this period caused marked point mutagens or simple deletions. Hence, to understand the PCBs-iron synergism more complex scenarios than point mutations or simple deletions must be invoked.