Iron-overloaded Rats Research Articles

Pattern of Iron Storage in the Rat Heart Following Iron Overloading with Trimethylhexanoyl-Ferrocene

Female Wistar rats with slight iron deficiency anemia were kept on a diet containing 0.5% trimethylhexanoyl (TMH)-ferrocene for up to 79 weeks. In the state of iron deficiency, the heart was free of light-microscopically detectable iron. After 7 weeks of the TMH-ferrocene diet, the first iron-positive granules appeared in perivascular macrophages. Further oral administration caused a progression of iron deposition in these cells, visible in the form of a granular staining but also as a diffuse iron staining of the cytoplasm. Accordingly, at the electron-microscopical level, the iron was stored partly as free ferritin molecules in the cytosol, and partly in lysosomes in the form of ferritin and/or hemosiderin. After 11 weeks, further iron-positive cells with relatively small dark-blue granules were found in the vicinity of capillaries, which could be identified as fibrocytes by means of electron microscopy. In addition, slight iron deposition occurred in the endothelial cells of the cardiac capillaries, likewise mainly in the form of small, uniform siderosomes. The myocytes showed no product of Perls' Prussian blue reaction during the whole period of investigation. From the 11th week onwards, discrete ferritin molecules were detected electron microscopically within lysosomes of these cells. Their amount increased slowly with progression of the TMH-ferrocene feeding period. Free ferritin molecules could be observed in the cytosol of fibrocytes, endothelial cells and myocytes in only very slight concentrations, whilst they were more plentiful in macrophages. In hereditary hemochromatosis and posttransfusional siderosis, the iron is found predominantly in myocytes and appears to cause cell damage, whilst this is not the case in experimental iron overload in rats.

Low dietary iron prevents free radical formation and heart pathology of copper-deficient rats fed fructose.

Two studies were conducted to determine whether hepatic iron overload in rats fed fructose plays a role in the exacerbation of the signs associated with copper deficiency. When fed the adequate iron diet (50 micrograms Fe/g), copper-deficient rats fed either fructose or starch exhibited hepatic iron overload of similar magnitude. However, only livers of copper-deficient rats fed fructose exhibited the presence of high peaks associated with an iron compound detected by electron spin resonance. In addition, only copper-deficient rats fed fructose developed anemia, pancreatic atrophy, and heart hypertrophy with histopathologic changes, and they died prematurely of heart-related abnormalities. Lowering dietary iron from 50 micrograms/g to 30 micrograms/g was not sufficient to protect the animals against the pathologic consequences of copper deficiency. In contrast, the consumption of a fructose diet inadequate in both copper (0.6 micrograms/g) and iron (17 micrograms/g) resulted in the reduction of hepatic iron, which in turn caused the amelioration of the deficiency, compared with rats fed the adequate iron (50 micrograms/g) diet. None of these rats developed pancreatic atrophy, none exhibited myocardial lesions, and none died of the deficiency. Electron spin resonance spectra of their livers did not show the presence of free radicals. The data suggest that hepatic iron overload plays a role in the exacerbation of copper deficiency only when fructose diets are consumed.

Metabolism of iron from (3,5,5-trimethylhexanoyl) ferrocene in rats: A dietary model for severe iron overload

The feeding of diets enriched with (3,5,5-trimethylhexanoyl)ferrocene (TMH-ferrocene) has been shown recently to produce a severe experimental iron overload in rats and has been considered as an adequate animal model for hereditary haemochromatosis in humans. We synthesized three 59Fe-labelled ferrocene compounds with different lipophilic characters ferrocene, TMH-ferrocene, and 1,1'-bis (3,5,5-trimethylhexanoyl)ferrocene [(TMH) 2-ferrocene] and studied the metabolism of iron from these compounds in comparison with the hydrophilic ferrous sulphate in rats with iron deficiency, and normal and increased iron stores. The bioavailability of iron from TMH-ferrocene (whole body retention, 48% from a 5 mg Fe dose) was twice as high as from ferrocene and six times higher than from (TMH) 2-ferrocene and ferrous sulphate. In contrast to the well-known iron salts (ferrous sulphate), the intestinal absorption of TMH-ferrocene iron was independent from the dose (1 or 5 mg Fe) and similar in iron-deficient and iron-loaded rats, indicating that the intestinal absorption of the TMH-ferrocene is not regulated by the body iron stores. After intestinal absorption, TMH-ferrocene iron in the portal blood is transported to the liver independently from transferrin. In contrast to absorbed ferrocene, iron from TMH-ferrocene is almost completely released from the hydrocarbon moiety within the liver. Depending on the body iron stores, TMH-ferrocene iron is then incorporated preferentially into haemoglobin (iron-deficient rats) or added to the iron stores in the liver (iron-loaded rats). A transient storage of the 59Fe-label in fat tissue was observed only from oral ferrocene but not from TMH-ferrocene. Due to the outstandingly high bioavailability of TMH-ferrocene, the chronic feeding of this compound resulted in a fast and progressive iron overload in rats (liver iron: 16.9 mg Fe g wet weight after 10 weeks of feeding a diet containing 0.5% TMH-ferrocene), and can be regarded as the best characterized and most useful animal model for severe hepatocellular iron overload in humans.

MR imaging of hepatic iron overload in rat.

To investigate the relationship of hepatic signal intensity and T2 with histologic grading in an animal model of oral iron overload and to determine the duration of feeding necessary to produce abnormalities detectable on magnetic resonance (MR) images, hepatic iron overload was induced in 12 rats by feeding them a diet supplemented with 4% carbonyl iron for 2-11 weeks. Iron overload seen on MR images was graded independently and blindly by two radiologists as normal, mild, moderate, or severe. The rats were killed, and histologic findings were graded blindly by four pathologists using a similar subjective scale. Hepatic T2 values were estimated from spin-echo images. In the rats with iron overload, intracellular iron deposition was noted on histologic studies. On MR images, hepatic signal intensity and T2 decreased after only 2 weeks of dietary iron overload, and both continued to decrease with longer duration of feeding. There was significant correlation between iron overload duration and changes on MR images and between MR images and histologic grading (r = .92, P = .0001 for both). The mean T2 of hepatic iron overload decreased with longer duration of feeding.

Noninvasive liver-iron quantification by computed tomography in iron-overloaded rats.

The benefit of computed tomography (CT) for the noninvasive determination of liver-iron concentration in human iron-overload diseases is a controversy in the literature. To study the sensitivity of CT for liver-iron quantification under experimental conditions, the authors measured single- and dual-energy CT numbers in vivo in the livers of iron-overloaded rats. Thirty-five rats were subjected to an iron-rich diet for various periods, from 1 to 20 weeks, then scanned by single- and dual-energy CT. CT absorption was correlated to liver-iron content, which was determined by wet ashing and spectrophotometry. Whereas a good correlation (r = 0.99 at 96 kV; r = 0.95 at 125 kV) between CT numbers and liver-iron concentration was found, CT was insensitive to low concentrations of iron. Dual-energy CT scanning results showed greater scattering in liver-iron quantification compared with single-energy CT. In rats, the sensitivity of single- and dual-energy CT is too low to quantify liver iron in the diagnostically most relevant region of mild liver siderosis (1-3 mg iron/g wet weight [w.wt]).

Evidence that an iron chelator regulates collagen synthesis by decreasing the stability of procollagen mRNA

Iron chelation has been shown previously to decrease collagen synthesis at a posttranslational level by inhibiting prolyl 4-hydroxylase, one of the key enzymes in collagen metabolism. On the other hand, recent in vivo studies of iron overload in rats suggest that iron could specifically activate collagen gene expression in liver tissues. These findings led us to investigate whether iron chelation might also affect collagen gene expression and posttranslational modification. Our data indicate that alpha,alpha'-dipyridyl, an iron chelator, at a concentration of 1 mmol/L, decreased steady-state levels of type I procollagen messenger RNA by 42% (p less than 0.001) without affecting beta-actin messenger RNA levels. Nuclear runoff studies demonstrated that transcription of the type I procollagen gene was unchanged by alpha,alpha'-dipyridyl. However, the turnover rate of type I procollagen messenger RNA was increased by 30%. This pretranslational inhibition of collagen synthesis was not due to decreased lipid peroxidation, because thiobarbituric acid-reactive substances were unchanged by alpha,alpha'-dipyridyl. However, cycloheximide totally abolished the effect, indicating that de novo protein synthesis was required.

Alterations in the structure, physicochemical properties, and pH of hepatocyte lysosomes in experimental iron overload.

While hemochromatosis is characterized by sequestration of iron-protein complexes in hepatocyte lysosomes, little is known about the effects of excess iron on these organelles. Therefore, we studied the effects of experimental iron overload on hepatocyte lysosomal structure, physicochemical properties, and function in rats fed carbonyl iron. A sixfold increase (P less than 0.0001) in hepatic iron and a fivefold increase in lysosomal iron (P less than 0.01) was observed after iron loading; as a result, hepatocyte lysosomes became enlarged and misshapen. These lysosomes displayed increased (P less than 0.0001) fragility; moreover, the fluidity of lysosomal membranes isolated from livers of iron-loaded rats was decreased (P less than 0.0003) as measured by fluorescence polarization. Malondialdehyde, an end product of lipid peroxidation, was increased by 73% (P less than 0.008) in lysosomal membranes isolated from livers of iron-overloaded rats. While amounts of several individual fatty acids in isolated lysosomal membranes were altered after iron overload, cholesterol/phospholipid ratios, lipid/protein ratios, double-bond index, and total saturated and unsaturated fatty acids remained unchanged. The pH of lysosomes in hepatocytes isolated from livers of iron-loaded rats and measured by digitized video microscopy was increased (control, 4.70 +/- 0.05; iron overload, 5.21 +/- 0.10; P less than 0.01). Our results demonstrate that experimental iron overload causes marked alterations in hepatocyte lysosomal morphology, an increase in lysosomal membrane fragility, a decrease in lysosomal membrane fluidity, and an increase in intralysosomal pH. Iron-catalyzed lipid peroxidation is likely the mechanism of these structural, physicochemical, and functional disturbances.

Transferrin Receptors and Selective Iron Deposition in Pancreatic B Cells of Iron‐overloaded Rats

Iron overload was produced in Wistar rats by repeated intraperitoneal injections of ferric nitrilotriacetate (Fe(3+)-NTA) for one to six months. Pancreatic tissues from these iron-overloaded rats and untreated controls were examined for insulin (for B cells), glucagon (for A cells), transferrin receptor (TfR), transferrin (Tf) and ferritin (Ft) using immunohistochemical methods, and for iron by histochemical Berlin blue staining. In the islets of iron-overloaded rats, increased Ft staining appeared prior to deposition of Berlin blue-stainable iron, and the staining intensity of Ft and iron was stronger in B cells than in A cells. In the islets of untreated control rats, the staining intensity of TfR was stronger in B cells than in A cells. TfR staining of the islets was weaker in iron-overloaded rats than in the controls. These findings suggest that 1) iron uptake by islet cells in vivo is regulated and mediated by TfR, 2) intracytoplasmic Ft transforms into stainable iron in iron-overloaded rats, and 3) predominance of TfR expression in B cells may result in selective deposition of iron and predispose B cells to damage and diabetes mellitus in iron-overloaded rats.

Chronic dietary iron overload in rats results in impaired calcium sequestration by hepatic mitochondria and microsomes 1, 2, and 3

The purpose of these experiments was to determine whether chronic dietary iron overload causes impairment of hepatic mitochondrial and/or microsomal calcium sequestration. Experimental iron overload was produced by feeding three groups of rats a chow diet supplemented with 3.0% (wt/wt) carbonyl iron for up to 8 weeks achieving graded increases in hepatic iron concentrations ranging from 1360 to 3170 μg/g. At low levels of iron overload, there were no changes in mitochondrial oxidative metabolism or calcium sequestration, whereas at moderate and high degrees of iron loading, both of these parameters were significantly reduced. In contrast, there were significant decreases in microsomal cytochrome P450 levels and microsomal calcium sequestration at all three levels of iron loading. These abnormalities occurred at hepatic iron concentrations at which the authors have previously found evidence of hepatic organelle lipid peroxidation. These alterations in organelle calcium sequestration may impair intracellular calcium homeostasis in the liver and contribute to subsequent cellular injury.

Liver Gene Expression During Chronic Dietary Iron Overload in Rats

To clarify the pathogenesis of hepatic iron toxicity, we investigated the effect of chronic dietary iron overload on the expression of several genes in rat liver. After 10 wk of iron treatment, when only minor histological features of liver damage were appreciable, the level of pro-alpha 2(I)-collagen mRNA was already higher than in control liver and increased further at 30 wk of treatment. Also, the relative amount of L ferritin subunit mRNA was enhanced early by iron load and was even more elevated at the latest time point considered, whereas neither H ferritin subunit nor transferrin mRNA levels were affected by iron treatment. In contrast, after chronic iron treatment, no variations were found in the steady-state level of mRNAs transcribed from liver-specific and preferentially expressed genes (albumin, alpha-fetoprotein, apolipoprotein A-1), growth-related genes (c-myc, c-Ha-ras and c-fos) and stress-induced genes (heat shock protein 70). These results suggest that chronic dietary iron overload in rats can specifically activate target genes in the liver (i.e., L ferritin and procollagen) in the absence of either histological signs of severe liver damage or alterations in differentiated liver functions.

Effect of iron overload on spontaneous and xenobiotic-induced lipid peroxidation in vivo.

To study the effect of iron-overload on hepatic lipid peroxidation, two rat models of haemochromatosis were employed: in the first model resembling secondary haemochromatosis, repeated i.p. injections with Fe-dextran led to an accumulation of Fe in Kupffer cells, while in the second model resembling hereditary haemochromatosis, iron was located mainly in periportal hepatocytes after feeding on a diet containing 3.5% Fe-fumarate for 3 weeks. In both models, total hepatic iron content was elevated four- to fivefold over controls. In vivo lipid peroxidation (ethane exhalation) was enhanced only in the second model, indicating that the hepatocytes are the main targets of Fe-induced lipid peroxidation. Low hepatotoxicity was observed in the second model. Additional treatment of the rats with hepatotoxic agents led to different results: with ethanol and bromobenzene, lipid peroxidation was only evident in both models of iron-overload, while paracetamol-induced lipid peroxidation was seen only in Fe-fumarate-fed rats. CCl4-induced lipid peroxidation was strongly enhanced in both models of haemochromatosis. Hepatotoxicity was enhanced by iron overload only in the case of CCl4-treated, Fe-fumarate-fed rats. The activities of phase I and phase II enzymes of xenobiotic metabolism were not markedly altered in livers of iron-overloaded rats. This implies that neither the bioactivation nor the detoxification of the agents studied were affected in experimental haemochromatosis.

Development of hexachlorobenzene porphyria in rats: Time sequence and relationship with lipid peroxidation

The relationship between the development of porphyria and free-radical formation induced by hexachlorobenzene was studied in iron-overloaded rats. The first sign of porphyria, an increase in porphyrins in the liver, was detected at day 22. Liver malondialdehyde was also increased at day 22. During the following weeks, liver porphyrins and malondialdehyde increased simultaneously, accompanied by a decrease in uroporphyrinogen decarboxylase activity and glucose-6-phosphate activity in liver, and a high excretion of porphyrins in the urine. In the rats given hexachlorobenzene, changes were detected in the pattern of lipids in the liver microsomes. In comparison with the controls, there were decreases in C20:4 and C22:5 fatty acids, whereas the fatty acid C20:3w6 was increased. In this study of hexachlorobenzene-induced liver damage there was no difference in the time course of the development of porphyria and that of lipid peroxidation.

Spontaneous and xenobiotic-induced in vivo-lipid peroxidation in iron-overloaded rats.

Iron ions play a major role in initiation and propagation reactions of enzymatic lipid peroxidation (LPO) (Aust et al 1985). Recently, it has been shown that chelation of endogenous iron by deferrioxamine, a specific high affinity iron chelator, resulted in an inhibition of acetaminophen- and CCl4-induced in vivo-lipid peroxidation (Younes and Siegers 1985). Similar results were obtained with bro-mobenzene (Casini et al 1987) and allyl alcohol (Jaeschke et al 1987), indicating that ionized iron is involved in xenobiotic-induced lipid peroxidation. On the other hand, simultaneous treatment with iron was shown to potentiate the prooxidative action of acetaminophen (Younes et al 1986), adriamycin (Muliawan et al 1986), CC14 (Muliawan and Kappus 1983), and ethanol (Nordmann et al 1987). In hemochromatosis (HC), excessive iron is progressively deposited in parenchymal cells of the liver, besides heart and endocrine glands (Powell 1975). Liver damage resulting from this condition, has been partly attributed to membrane lipid peroxidation of mitochondria and microsomes with subsequent cell injury and cell death (Bacon et al 1983, 1985). In this study, the effect of experimental iron-overload on lipid peroxidation induced by acetaminophen, ethanol, bromobenzene and CC14 was investigated using two rat models resembling hereditary and secondary HC, respectively.

Quantitative analysis of immunogold labelling for ferritin in liver from control and iron-overloaded rats.

The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry. Immunoreactive ferritin was present in the cytosol, cytosolic clusters and lysosomes of normal hepatocytes. After iron-loading, the labelling density increased over tenfold in parenchymal cell cytosol with a smaller increase in Kupffer cells. Ferritin clusters contained substantially more immunoreactive ferritin than equivalent areas of lysosomes or cytosol. Analysis of the labelling density in hepatocyte lysosomes showed that, despite a striking increase in iron content, one-quarter of the lysosomes showed less immunolabelled ferritin than the cytosol. The existence of a wide range of ferritin labelling densities in the lysosomes with a large proportion unlabelled suggests that the ferritin protein shell is not degraded at a significant rate either in the cytosol or in clusters but only after incorporation into lysosomes.

Quantitative Analysis of Immunogold Labelling for Ferritin in Liver of Control and Iron-Overloaded Rats

The distribution of ferritin antigenicity in control and iron-loaded rat hepatocytes was investigated with an immunogold-ferritin antibody technique. Antibody to horse spleen ferritin showed immunoreactivity as determined by dot blotting with immunogold/silver staining with purified rat liver ferritin but not with rat haemosiderin. The initial site of ferritin degradation was studied by analysing the density of gold labelling in the cytosol and lysosomes in combination with pre-embedding acid phosphatase cytochemistry.

Involvement of iron and iron-catalyzed free radical production in ethanol metabolism and toxicity.

Lipoperoxidation, a degradative process of membranous polyunsaturated fatty acids, has been suggested to represent an important mechanism in the pathogenesis of ethanol toxicity on the liver and possibly also on the brain. Catalysis by transition metals, especially iron, is involved in the biosynthesis of free radicals contributing to lipid peroxidation. Although the exact nature of the redox-active iron implicated in this catalysis is still unknown, it has been well established that lipid peroxidation can be prevented in vitro by iron chelators such as desferrioxamine. Deprivation of redox-active iron through desferrioxamine inhibits by about 50% the microsomal oxidation of ethanol in vitro and reduces very significantly in vivo the overall ethanol elimination rate in rats. Administration of desferrioxamine together with ethanol also reduces the ethanol-induced disturbances in the antioxidant defense mechanisms of the hepatocyte. It also reduces in mice both the severity of physical dependence on ethanol and lethality following the acute administration of a narcotic dose of ethanol. Chronic overloading of rats with iron results, on the opposite, in an increased rate of ethanol elimination, although alcohol dehydrogenase and catalase activities are reduced and cytochrome P-450 depleted in the liver of such iron-overloaded animals. The magnitude of the ethanol-induced increase in lipid peroxidation and decrease in the major membranous antioxidant, alpha-tocopherol, is exacerbated in iron-overloaded rats. Several disturbances of iron metabolism have been reported in human alcoholics. Their contribution to ethanol toxicity appears very likely in the case of hepatic siderosis associated with alcohol abuse. Ethanol could however disturb iron metabolism even in the absence of gross abnormalities of the total iron stores. It is suggested that ethanol intoxication could increase cellular redox-active iron, thus contributing to an enhanced steady-state concentration of reactive-free radicals. This oxidative stress would lead to lipoperoxidative damage and cellular injury.

Increased resistance of peroxisome proliferator-induced hepatic lesions to iron overload in rats

Altered areas (AA), neoplastic nodules (NN) and hepatocellular carcinomas (HCC) induced by chronic administration of ciprofibrate and Wy-14,643 were examined for iron accumulation after systemic iron overload. Eighty percent of AA, 90% NN and 100% HCC showed increased resistance to iron accumulation. However, marked heterogeneity was observed in the amount of iron from area to area within the same lesion with some cells containing no iron, while others showing blue reaction to iron. These findings indicate, that putative preneoplastic and neoplastic hepatic lesions induced by peroxi-some proliferators exclude iron in a fashion similar to that of hepatic nodules and carcinomas induced by DNA damaging carcinogens.

Effects of iron deficiency and chronic iron overloading on mitochondrial heme biosynthetic enzymes in rat liver

The effects of iron deficiency and iron overloading on the mitochondrial enzymes involved in heme synthesis were studied in rat livers. The in vitro activities of several of the enzymes in this pathway were differentially influenced by the in vivo iron status of the animals. δ-Aminolevulinic acid synthase was slightly increased in iron-overloaded animals, but remained normal in iron-deficient animals (0.58 ± 0.09, 0.91 ± 0.19 and 0.61 ± 0.12 nmol δ-aminolevulinic acid/mg per h). Copro- and protoporphyrinogen oxidase activities were increased (20 and 60% above controls) in iron-deficient animals. In contrast, coproporphyrinogen oxidase was decreased by 20%, while protoporphyrinogen oxidase remained unchanged in iron-overloaded rats. These variations of activities were not due to changes in the affinity of these enzymes toward their substrates, as coporphyrinogen had the same Km in each case (0.62 ± 0.05 M) as did protoporphyrinogen (0.22 ± 0.035 M). Thus, the Km did not vary with the treatment received by the animals. Ferrochelatase activity was measured by both the pyridine hemochromogen method and by measurement of zinc protoporphyrin with endogenous zinc as substrate. In all cases, ferrochelatase was found to be able to synthesize zinc protoporphyrin with endogenous zinc as substrate. However, the apparent Km of zinc chelatase for protoporphyrin was significantly different in the three groups of animals with Km,appProto = 2.4 ± 0.1 10−7, 4 ± 0.3 10−7 and 9.10 ± 0.05 10−7 M in iron-overloaded, control and iron-deficient animals, respectively. When ferrochelatase activity was measured by pyridine hemochromogen, identical results were observed in iron-deficient and control animals but decreased by 45% in iron-overloaded animals. The mitochondrial heme content was also decreased by 40% in iron-overloaded rats but unchanged in either iron-deficient or control rats.

Renal impairment in experimental hemochromatosis in rats.

Parenteral administration of iron as the complex with nitrilotriacetate resulted in moderate iron deposition in the renal cortex along with heavy deposition of iron in liver, heart, and exocrine pancreas. Iron treated rats exhibited slight but significant changes in serum chemistries consistent with mild renal insufficiency: increased chloride and creatinine and decreased sodium, calcium and CO2 content as a group with isolated increases in urea nitrogen (2/9) and phosphorus (3/9). Serum albumin was decreased and the normal correlation of serum calcium with serum albumin was abolished by iron overload. Although food intake/g body weight was similar in the two groups, both water intake and urine output relative to solute load were increased in iron-overloaded rats. We conclude that the renal insufficiency occasionally reported in human hemochromatosis may reflect a direct nephrotoxic action of iron.

Control of delta-aminolaevulinate synthase and haem oxygenase in chronic-iron-overloaded rats.

The hepatic porphyrias are inborn errors of porphyrin and haem biosynthesis characterized biochemically by excessive excretion of delta-aminolaevulinate (ALA), porphobilinogen and other intermediates in haem synthesis. Clinical evidence has implicated iron in the pathogenesis of several types of genetically transmitted diseases. We investigated the role of iron in haem metabolism as well as its relationship to drug-mediated induction of ALA synthase and haem oxygenase in acute and chronic iron overload. Acute iron overload in rats resulted in a marked increase in hepatic haem oxygenase that was associated with a decrease in cytochrome P-450 and an increase in ALA synthase activity. Aminopyrine N-demethylase and aniline hydroxylase activities, which are dependent on the concentration of cytochrome P-450, were also decreased. In contrast, in chronic-iron-overloaded rats, there was an adaptive increase in haem oxygenase activity and an increase in ALA synthase that was associated with normal concentrations of microsomal haem and cytochrome P-450. The induction of ALA synthase in chronic iron overload was enhanced by phenobarbital and allylisopropylacetamide, in spite of the fact that these agents did not increase haem oxygenase activity. Small doses of Co2+ were potent inducers of the haem oxygenase in chronic-iron-overloaded, but not in control, animals. We conclude that increased hepatic cellular iron may predispose certain enzymes of haem synthesis to induction by exogenous agents and thereby affect drug-metabolizing enzyme activities.