Biliary excretion of green pigments produced by norethindrone in the rat

Bile was a major route for the excretion of green pigments produced in the liver following treatment of rats with the contraceptive steroid norethindrone (100 mg/kg, i.p.). Only very low concentrations were detected in the plasma and none in extracts of urine. No green pigments of this type were detected in control rat bile. Separation of the biliary green pigments following esterification with methanol/H₂SO₄ by HPLC or TLC showed the presence of two major components. These co-chromatographed with two of the major components present in the liver of rats given norethindrone. Biliary green pigments had a similar visible spectrum to those extracted from liver with the Soret absorption maximum in CHCl₃ at 417 nm. The time course for the excretion of green pigments in the bile showed maximum concentrations were reached 6 hr after dosing followed by a more gradual decline over the next 18 hr. The total concentration of green pigments excreted into the bile in the 24 hr following dosing of rats with norethindrone was greater than the initial concentration of cytochrome P-450 in the liver. Pretreatment of rats with cycloheximide (1.5 mg/kg) prior to norethindrone reduced the concentration of green pigments in the bile over an 8 hr period by about 40% but caused no accumulation of these compounds in the liver. This suggested protein synthesis may be necessary for the continued formation of green pigments. When rats were dosed with [⁵⁹Fe]FeCl₃ prior to norethindrone, biliary extracts contained ⁵⁹Fe radioactivity. This was significantly higher than in control rats given only ⁵⁹Fe. TLC of green pigments from the bile of norethindrone dosed rats, esterified under neutral pH conditions, showed ⁵⁹Fe radioactivity associated with a poorly defined green component (R_f 0.23) which did not fluoresce red under u.v. light. Results suggested that green pigments in the bile still contained chelated iron. Green pigments excreted into the bile following dosing with norethindrone were found in the faeces, the majority within 48 hr of dosing. The amounts excreted suggested they were largely resistant to attack of enzymes from the gut and gut flora.     

Species differences in hepatic microsomal drug-metabolizing enzymes

The activity of some metabolizing enzymes was assessed in the liver microsomes of Acomys cahirinus, mice and rats. The enzymatic studies were followed by the determination of cerebral level of apomorphine (APO), imipramine (IMI) and its metabolite desipramine (DMI) of animals treated with a single dose of APO or IMI. It was found that the level of cytochrome P-450 and the activity of IMI demethylase and glucuronyltransferase in the liver microsomes of rats was significantly higher than those in the liver microsomes of Acomys and mice. The brain levels of APO, IMI and DMI were different in investigated species and IMI and DMI levels in the brain of Acomys, mice and rats corresponded to the activity of IMI demethylase in the liver microsomes of these species.     

Species differences in the hepatic and intestinal metabolism of cyclosporine

1. Cyclosporin A (cyclosporine, CSA) is an immunosuppressive drug with a narrow therapeutic index. In the present study the metabolism of CSA was investigated in the liver and small intestinal microsomes obtained from rat, hamster, rabbit, dog, baboon and man by measuring the disappearance of CSA and the formation of the principal metabolites of CSA, namely hydroxylated and N-demethylated CSA. 2. CSA was metabolized at a very slow rate (2-8% metabolism in 30 min) in rat liver microsomes whereas microsomes from dog livers were very efficient (70-100% metabolism in 30 min) in metabolizing CSA. Hydroxylation and N-demethylation accounted for most of the CSA metabolized in all the species tested. 3. Microsomes from the small intestine produced qualitatively a similar metabolic profile as compared with the microsomes from the liver, but at a slower rate in all the species tested. The relative importance of the different metabolic pathways, however, differed between species. 4. This study points to the importance of recognizing the similarities and the differences in the metabolism of CSA in different species when data from animal studies are extrapolated to man.     

Species differences in the hepatic and intestinal metabolism of cyclosporine

1. Cyclosporin A (cyclosporine, CSA) is an immunosuppressive drug with a narrow therapeutic index. In the present study the metabolism of CSA was investigated in the liver and small intestinal microsomes obtained from rat, hamster, rabbit, dog, baboon and man by measuring the disappearance of CSA and the formation of the principal metabolites of CSA, namely hydroxylated and N-demethylated CSA. 2. CSA was metabolized at a very slow rate (2-8% metabolism in 30 min) in rat liver microsomes whereas microsomes from dog livers were very efficient (70-100% metabolism in 30 min) in metabolizing CSA. Hydroxylation and N-demethylation accounted for most of the CSA metabolized in all the species tested. 3. Microsomes from the small intestine produced qualitatively a similar metabolic profile as compared with the microsomes from the liver, but at a slower rate in allthe species tested. The relative importance of the different metabolic pathways, however, differed between species. 4. This study pointsto the importance of recognizing the similarities andthe differences in the metabolism of CSA in different species when data from animal studies are extrapolated to man.     

Species differences in the hepatic microsomal enzyme metabolism of the pyrrolizidine alkaloids

Species differences in pyrrolic metabolites and senecionine (SN) N-oxide formation among eight animal species (sheep, cattle, gerbils, rabbits, hamsters, Japanese quail, chickens, rats) varying in susceptibility to pyrrolizidine alkaloid (PA) intoxication were measured in vitro by hepatic microsomal incubations. The results suggested that there is not a strong correlation between the production of pyrrolic metabolites and susceptibility of animals to PA toxicity. The rate of PA activation in hamsters, a resistant species, measured by formation of (±)6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) far exceeded the rate of SN N-oxide formation (detoxification) (DHP/N-oxide=2.29). In contrast, SN N-oxide was the major metabolite in sheep, another resistant species, with much lower production of DHP (DHP/N-oxide=0.26). The roles of cytochrome P450s and flavin-containing monooxygenases (FMO) in bioactivation and detoxification of pyrrolizidine alkaloids (PA) were studied in vitro using sheep and hamster hepatic microsomes. Chemical and immunochemical inhibition data suggested that the conversion of SN to DHP is catalyzed mainly by cytochrome P450s (68–82%), whereas the formation of SN N-oxide is carried out largely by FMO (55–71%). There also appeared to be a high rate of glutathione–DHP conjugation in hamster (63%) and sheep (79%) liver microsomal incubation mixtures. Therefore, low rates of pyrrole metabolite production coupled with glutathione conjugation in sheep may explain the resistance of sheep to SN, whereas the high rate of GSH-DHP conjugation may be one of the factors contributing to the resistance of hamsters to intoxication by this PA.     

Species differences in the formation of butadiene monoxide from 1,3-butadiene

When 1,3-butadiene is incubated with liver postmitochondrial fractions from mouse, rat, monkey or man and a NADPH-regenerating system, the formation rate of butadiene monoxide is different in the four species. With the exception of the rhesus monkey, the amount of epoxide is proportional to the monooxygenase activity. The sequence of epoxide formation is B6C3F₁ mouse, Sprague Dawley rat, man, rhesus monkey. The ratio between mouse and monkey was about 71. When 1,3-butadiene is incubated with homogenates from lung tissue, only tissues from mouse and rat produce measurable butadiene monoxide concentrations. The monooxygenase activity in lung tissue of the mouse was only 1/30 that in mouse liver. By contrast, lung tissue formed epoxide concentrations comparable to those formed by liver tissue, whereas monkey and human lung tissue did not produce any measurable levels of butadiene monoxide. The data might suggest that the results of recent rodent inhalation studies with 1,3-butadiene could not automatically be extrapolated to man.     

Induction of hepatic metallothionein following administration of urethane

Induction of hepatic metallothionein (MT) by urethane (ethyl carbamate) was characterized. Male CF-1 mice were treated with urethane (0, 0.5, 1.0, 1.5, and 2 g/kg; ip) and 18 hr later hepatic MT concentrations were determined with the Cd-hemoglobin radioassay. Urethane (1 g/kg and higher) significantly increased hepatic MT levels, resulting in a 14-fold increase after 2 g/kg. Time-course experiments indicated that MT levels were increased significantly at 6 hr after administration of urethane (1.5 g/kg) and reached a maximum between 12 and 24 hr. Gel filtration, anion-exchange chromatography, and ultraviolet spectral analysis were used to characterize the protein induced by urethane. Pretreatment with actinomycin-D prevented induction of MT by urethane. Administration of equimolar dosages (20 mmol/kg) of urethane, N-hydroxyurethane, and methyl carbamate indicated that urethane and N-hydroxyurethane induce MT but that methyl carbamate does not. MT induction was also not observed with other commonly used anesthetics (pentobarbital and phenobarbital). In conclusion, urethane induces hepatic MT but this effect is not related to its anesthetic action, nor is it a common property of all carbamates.     

Induction of hepatic metallothionein following 5-azacytidine administration

Exposure of cells in culture to the pyrimidine analog, 5-azacytidine (AZA-C), stimulates the expression of the metallothionein (MT) gene. Therefore this study was performed in an attempt to extend this observation to a whole animal system. Young Wistar rats (approximately 30 to 35 days old) were administered AZA-C 24 and 16 hr (50 mg AZA-C/kg, ip, each time) prior to the determination of hepatic MT by the Cd-Hem method. Such treatment resulted in an approximately fivefold increase in the concentrations of hepatic MT in cytosol obtained from both male and female rats. Gel-filtration and anion-exchange chromatography, as well as uv spectral analysis, confirmed the presence of MT in the livers of AZA-C-treated animals. A single dose of AZA-C (65 mg/kg, ip) produced increases in hepatic MT concentrations 8 hr after dosing that were still elevated at 48 hr. Dose-response studies indicated hepatic MT concentrations were increased 24 hr after the administration of 50, 60, and 65 mg AZA-C/kg but were not altered by doses of 30 or 40 mg/kg. Actinomycin-D pretreatment (1.25 mg/kg, ip) 30 min prior to AZA-C (80 mg/kg, ip) prevented the subsequent increases in hepatic MT concentrations. These data indicate that treatment with AZA-C produces an in vivo induction of hepatic MT synthesis that appears to be more persistent in nature than other nonmetallic inducing agents.     

Induction of hepatic metallothionein in the immature rat following administration of cadmium

Hepatic metallothionein (MT) levels, as measured indirectly by total metal-binding capacity, are approximately 8.5-fold higher in the 7-day-old rat than in the 28-day-old rat where levels are barely detectable. To stydy how the presence of MT might influence the toxicity of cadmium, a single subcutaneous injection of cadmium chloride was administered at dose levels of 0, 1, 2, 3, and 6 mg Cd/kg body weight, 48 hr prior to sacrifice on Day 7 or Day 28. In both the 7- and 28-day-old groups, there was a dose-related increase in the amount of cadmium bound to MT. There were no significant age-related differences in the amount of cadmium bound to MT at the various doses, with the exception of the 6 mg/kg dose, where 7 day levels were higher. The 28-day-old rats responded to cadmium exposure with induction of MT and subsequent binding of both cadmium and zinc at all doses. The 7-day-old appeared to have sufficient levels of MT to handle cadmium doses at or below 3 mg/kg without induction; however, at the 6 mg/kg dose induction was observed. Despite the presence and inducibility of hepatic MT at Day 7, 30% of the rats treated with 6 mg Cd/kg died within 48 hr of exposure compared with only 4% at Day 28.     

Species differences in stimulation of intestinal and hepatic microsomal mixed-function oxidase enzymes

The effect of intraperitoneal (i.p.) administration of phenobarbital (PB) or 3-methylchol-anthrene (3-MC) on some mixed-function oxidase (MFO) enzymes was studied in small intestine and liver of male rats, mice, guinea pigs and rabbits. PB treatment enhanced intestinal and 7-ethoxycoumarin deethylase activities in the mouse and rat, whereas benzo[a]pyrene hydroxylase (AHH) activity was increased only in the mouse. Ethylmorphine demethylase and aniline hydroxylase activities in small intestine were not stimulated by PB in any species. Administration of 3-MC increased the activity of intestinal AHH in rat, mouse and guinea pig, but intestinal 7-ethoxycoumarin deethylase activity was elevated only in the rat. The guinea pig and mouse intestinal ethoxycoumarin deethylase activity was inhibited by 3-MC treatment. None of the enzymes tested in rabbit intestine was induced by PB or 3-MC. The hepatic activities of ethylmorphine demethylase, aniline hydroxylase, 7-ethoxycoumarin deethylase and AHH, and the cytochrome P-450 content were increased by PB in all species. In contrast, 3-MC enhanced hepatic aniline hydroxylase and AHH activities in rats, mice and guinea pigs, and hepatic 7-ethoxycoumarin deethylase activity in mice and rats. In rabbits, these hepatic enzymes were inhibited by 3-MC pretreatment. The hepatic cytochrome P-450 absorption spectra was shifted to 448 nm in all species. These results suggest that there are differences in induction of intestinal and hepatic MFO enzymes which are influenced by the type of inducing agent, substrate and animal species used.     

Transient induction of hepatic metallothionein following oral ethanol administration

Chronic ethanol ingestion has been associated with alterations of zinc homeostasis. Various treatments that alter zinc disposition induce hepatic metallothionein (MT). Therefore, this study was performed to determine the effect of acute ethanol exposure on hepatic MT levels. Adult male CF-1 mice were administered ethanol intragastrically and their hepatic MT was quantified at various times thereafter by the Cd-radioassay method. Ethanol (5 g/kg, ig) produced significant increases in hepatic MT as early as 4 hr after dosing. Maximal hepatic MT concentrations (19-fold increase) were observed 24 hr after ethanol and returned to control concentrations by 48 hr. Hepatic MT levels were increased 24 hr after 5 or 7 g ethanol/kg but were not altered by 1, 2, or 3 g/kg. Elevations in pancreatic MT, but not in renal or intestinal MT, also occurred 24 hr after ethanol (5 g/kg). Actinomycin D (1.25 mg/kg, ip) prevented the increase in hepatic MT produced by ethanol, whereas inhibition of ethanol oxidation by pyrazole (150 mg/kg, ip) did not prevent the induction of hepatic MT. Gel filtration chromatography and uv spectral analysis confirmed the presence of MT in the livers of ethanol-treated mice. These data show that acute ethanol administration produces a marked elevation of hepatic MT that is transient.     

Species and sex differences in the metabolism of a chlorinated epoxide by hepatic microsomal enzymes

A dimethylated chlorocyclodiene epoxide (DME), proposed as a suitable substrate for the study of microsomal monooxygenases in rats, yields two major metabolites, M1 and M2, when incubated with NADPH-supplemented hepatic microsomes from adult male rats but only one when incubated with those from adult female rats. Similar microsomes from male and from female mice, pigeons and chickens produce both metabolites. The ratio of the amounts of the two metabolites formed varies with the species but no sex difference within any one of these species has been demonstrated. The rate of metabolism of DME varies both with sex and with species: hepatic microsomes from female mice metabolise it faster than do those from males whereas hepatic microsomes from male rats metabolise it faster than do those from females. Hepatic microsomes from both sexes of the domestic chicken metabolise DME slowly, converting it predominantly to metabolite M2. The use of [¹⁴C]DME has established that the apparent absence of metabolite M2 from the incubates containing liver preparations from female rats is not attributable to low metabolism nor is it caused by metabolite M2 being converted to other metabolites as soon as it is formed. Little radioactivity remains behind in the aqueous phase after hexane extraction of incubation media containing [¹⁴C]DME and female rat liver microsomes. Florisil column separation of the components of the hexane extracts indicates that little radioactivity is present in eluate fractions other than those showing a g.l.c. peak for DME or for metabolite Ml or for metabolite M2. Hepatic microsomes from young rats of both sexes metabolise DME at similar rates until they approach 28 days of age. Thereafter, the rate in those from male rats increases whereas that from those from female rats falls. The hepatic microsomes from male rats castrated when seven days of age metabolise DME in adulthood at the same rate as those from intact female rats. The relevance of these observations to the proposed use of DME is discussed.     

Destruction of cytochrome P-450 and formation of green pigments by contraceptive steroids in rat hepatocyte suspensions

The contraceptive steroid norethindrone caused a rapid time and dose-dependent loss of cytochrome P-450 from rat hepatocytes in suspension cultures. Up to 30% of this cytochrome was lost in the first 5 min of incubation; longer incubations resulted in little further loss even though not all the steroid was metabolised and the cells remained viable. Such cultures were used to investigate the formation of N-alkylated porphyrins (green pigments) which could be extracted from cell incubation mixtures following exposure to norethindrone and separation by HPLC or TLC. The number of N-alkylated porphyrins formed was dependent both on the time of incubation and the concentration of steroid. After 1 min, 1 major green pigment (GP1) was resolved using either high (0.3 mM) or low (0.03 mM) norethindrone concentrations. With longer incubation times (60 min), at high steroid concentrations, only one additional polar adduct (GP2) was formed. At lower steroid levels, 3 more polar components (GP2, 3 and 4) were seen. As judged by HPLC or TLC, GP1 corresponds to the pigment formed in microsomal preparations incubated with norethindrone in vitro, while GP2, 3 and 4 correspond to the pigments extracted from the livers of rats administered this steroid in vivo. Pretreatment of rats with either phenobarbitone or 3-methylcholanthrene induced cytochrome P-450s. Relative to controls, phenobarbitone pretreatment also resulted in a greater accumulation of green pigments in hepatocytes incubated with norethindrone, the more polar forms of green pigments (GP3 and 4), showing a disproportionate increase in concentration. The mixed function oxidase inhibitor SKF 525-A or high concentrations of steroid not containing an ethynyl function, e.g. norethandrolone, when added to cell cultures containing norethindrone, preferentially inhibited the formation of GP3 and 4. When purified green pigments were added to cell incubation mixtures in the absence of norethindrone, preferentially inhibited the formation of GP3 and 4. When purified green pigments were added to cell incubation mixtures in the absence of norethindrone, no interconversion of one form to another could be demonstrated. The results suggest that the more polar norethindrone-protoporphyrin IX adducts (GP2, 3 and 4) arise as a result of metabolic modification of norethindrone rather than the protoporphyrin IX moiety, either prior to or after activation of the ethynyl function. The formation of several green pigment components in hepatocyte suspensions was not unique to norethindrone but occurred with a number of other 17-ethynyl-substituted steroids.     

Induction of hepatic metallothionein in mouse liver following administration of chelating agents

Chelating agents commonly used in therapy of heavy metal intoxication alter the levels of essential metals in liver, kidneys, and serum. Induction of metallothionein synthesis in liver occurs following exposure to a variety of chemical and environmental insults and, in some cases, has been attributed to enhanced hepatic uptake of zinc. Therefore, the effect of acute exposure to seven common metal chelators on the concentration of metallothionein in liver was investigated. Adult male Swiss Webster mice were injected intraperitoneally with the chelators and hepatic metallothionein was quantified by the cadmium radioassay. Ethylenediaminetetraacetic acid (EDTA) produced a 5- to 6-fold increase in hepatic metallothionein 24 hr after injection of 0.75 to 3.0 g/kg. No significant increase in hepatic MT was observed until 12 hr following injection of EDTA (1.5 g/kg, ip). Maximal levels were reached between 12 and 48 hr following EDTA injection. Cadmium, a known inducer of hepatic metallothionein, produced a 15-fold increase in the concentration of MT in liver 24 hr following injection. By comparison, 2,3-dimercaptopropanol and diethyldithiocarbamate produced a 9-fold and 13-fold increase in hepatic metallothionein levels, respectively, 24 hr following injection. A 4- to 6-fold increase in metallothionein was observed 24 hr following injection of 2,3-dimercaptosuccinic acid, D,L-penicillamine, diethylenetriaminepentaacetic acid, and EDTA, while nitrilotriacetic acid elevated hepatic metallothionein levels by 2-fold. Alterations in the concentration of hepatic metallothionein by chelators may have implications for their efficacy in the treatment of cadmium intoxication.     

Proliferation of hepatic peroxisomes in rats following the intake of green or black tea.

Rats maintained on green, black or decaffeinated black tea (2.5%, w/v) as their sole drinking fluid displayed higher hepatic CN- insensitive palmitoyl CoA oxidase activity than controls; the extent of increase was similar with the three types of tea. Morphological examination of the liver using electron microscopy revealed an increase in the number of peroxisomes in the tea-treated animals. The same treatment of the animals with green and black tea resulted in a similar rise in hepatic microsomal lauric acid hydroxylation. Analysis by HPLC of the aqueous tea extracts employed in the current study showed that the total flavanol content of the green variety was much higher than the black varieties, and confirmed the absence of caffeine in the decaffeinated black tea. It may be concluded from the present studies that neither caffeine nor flavanoids are likely to be responsible for the proliferation of peroxisomes observed in rats treated with tea.     

Enhancement of hepatic microsomal drug metabolism in vitro following ethanol administration.

1. Administration of ethanol intraperitoneally at low dosages (10-25 mg/kg) to rats stimulates hepatic microsomal mixed-function oxidase activity in vitro. 2. Pretreatment with ethanol administered orally has no effect on in vivo drug metabolism as measured by pentobarbitone plasma half-life and has no effect on the excretion of ascorbic acid. Ethanol administration does not enhance its own binding to cytochrome P-450. 3. These observations suggest that the administration of ethanol, at moderate dosage, does not give rise to induction of hepatic cytochrome P-450. 4. Unwashed hepatic microsomes are contaminated with alcohol dehydrogenase, but pretreatment with ethanol does not increase microsomal generation of NADH. 5. Pretreatment with ethanol has no stimulatory effect on NADH-NADP+ transhydrogenation. 6. The stimulation of hepatic drug metabolism in vitro following administration of ethanol is not due to increased cytochrome P-450 nor to increased NADPH, per se, but appears to result from an increase in the activity of NADPH-cytochrome c reductase.     

Species differences in the substrate specificity of hepatic cytochrome P-448 from polycyclic hydrocarbon-treated animals.

The in vitro effects of α-naphthoflavone on four hepatic mono-oxygenase activities associated with aromatic hydrocarbon responsiveness in the mouse (aryl hydrocarbon hydroxylase, 2-acetylamino-fluorene N-hydroxylase, biphenyl 2-hydroxylase, and biphenyl 4-hydroxylase) were investigated before and after methylcholanthrene treatment of C57BL/6N and DBA/2N mice, rats, hamsters, guinea pigs and rabbits. The electrophoretic pattern of cytochrome P-450 subunits and reduced CO-hemoprotein difference spectra of the microsomal fractions were also studied. Pretreatment of animals with methylcholanthrene caused: (1) a 1.5 to 2 mm hypsochromic shift in the Soret peak of the reduced hemoprotein-CO complexes in liver microsomes from a C57BL/6N mouse, rat, hamster and rabbit; a 0.5-nm hypsochromic shift in the guinea pig and no shift in the DBA/2N mouse; and (2) an increase in cytochrome P-450 apoproteins of the following molecular weights on sodium dodecyl sulfate-polyaerylamide gel electrophoresis: 54,000 and 55,000 in the C57BL/6N mouse; 48,000, 54,000 and 55,000 in the rat; 49,000 and 54,000 in the hamster; and 54,000 and 57,000 in the rabbit; a small increase in the 54,000 band was seen in the DBA/2N mouse and no increase in the guinea pig. In vitro addition of α-naphthoflavone selectively inhibited all four mono-oxygenase activities from the methylcholanthrene-treated C57BL/6N mouse, rat and hamster; 2-acetylaminofluorene N-hydroxylase and biphenyl 4-hydroxylase activities in the rabbit; and aryl hydrocarbon hydroxylase, 2-acetylaminofluorene N-hydroxylase and biphenyl 4-hydroxylase activities in the guinea pig. The addition of α-naphthoflavone enhanced the activities of aryl hydrocarbon hydroxylase and biphenyl 2-hydroxylase in liver microsomes from both control and methylcholanthrenetreated rabbits, but only biphenyl 2-hydroxylase activity was increased in the guinea pig: the activitity of 2-acetylaminofluorene N-hydroxylase was increased in both control and methylcholan-threne-treated DBA/2M mice, but only in the control C57BL/6N mouse. These data indicate that hepatic cytochrome P-448 is composed of multiple cytochromes, which differ among animal species, each catalyzing different mono-oxygenase activities.