The Use of Uninduced Rat Livfr S-9 to Supplement BALB/3T3 Cells in the In Vitro Transformation Assay

ABSTRACT

Because of limited inherent capacity to metabolize chemicals to their reactive form, the BALB/3T3 transformaticn assay using clone A31–1–1 cells requires metabolic supplementation with rodent liver homogenate preparations (S-9). Activation by 8–9 is limited, however, by its cytotoxicity to the cells, thus necessitating a reduction in treatment time from the usual 24–72 to 1–4 hr. With cyclophosphamide (CP) as a test chemical, we were able to increase the treatment time to 24 hr by using lower concentrations of cofactors and S-9 prepared from the livers of untreated rats. Statjstically significant increases in transformed foci were induced in cultures treated with 50 or 100 vg CP/ml in the presence of 100 or 200 vg of uninduced rat liver S-9/ml and 76 or 380 μg each of NAPH, NADP, NADPE, and glucose-6-phosphate per ml of incubation medium.     

S-9 metabolic activation enhances aflatoxin-mediated transformation of C3H/10T1/2 cells

The use of a rat liver S-9 metabolic activation system to enhance aflatoxin B1-mediated transformation of C3H/10T1/2 cells was studied. Under conditions of metabolic activation, the cytotoxicity of Aflatoxin B1 (AFB) was increased approximately 10-fold over that observed in the absence of activation. Similarly, activation increased the transformation of these cells treated with 1 microgram/ml AFB1 greater than 10-fold when cells were treated in the absence of activation with 1 microgram/ml AFB1 for 3 hr, and four-to-fivefold over the transformation observed when cells were treated for 48 hr with 1 microgram/ml AFB1. This same activation procedure also induced the transformation of these cells treated with 10 and 20 micrograms/ml cyclophosphamide in the absence of activation. The use of S-9 metabolic activation greatly increased the sensitivity of AFB1-mediated transformation of C3H/10T1/2 cells, and should expand the range of chemical carcinogens, requiring metabolic activation, in particular mycotoxins related to AFB1, that can be effectively detected and studied in the C3H/10T1/2 cell transformation assay.     

Role of cyclic AMP in the inhibition of mouse hepatocyte intercellular communication by liver tumor promoters

The liver tumor promoters phenobarbital (PB) (20-500 micrograms/ml) and 1,1-bis(4-chlorophenyl)-2,2,2-trichloroethane (DDT) (1-10 micrograms/ml) inhibited intercellular communication between primary cultured B6C3F1 mouse hepatocytes after 8 hr of treatment. Intercellular communication was detected autoradiographically as the passage and incorporation of [5-3H]uridine nucleotides from prelabeled donor hepatocytes to recipient hepatocytes. The addition of either dibutyryl cyclic AMP (N6,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) (0.001-0.1 mM) or caffeine (0.01-1 mM) decreased or completely abolished the inhibitory effects of PB and DDT on intercellular communication. Cyclic AMP (adenosine 3':5'-cyclic monophosphate; cAMP) in primary cultured mouse hepatocytes was measured by radioimmunoassay. Cyclic AMP in nontreated, freshly plated cultures declined from 4.2 +/- 0.7 pmol/mg protein after 1 hr in culture to 2.4 +/- 0.5 pmol/mg protein after 8 hr in culture. Phenobarbital at 250 and 500 micrograms/ml significantly decreased cyclic AMP below control values after 1 hr of treatment. However, no difference in the amount of cyclic AMP was detected between control and PB-treated cultures after 2, 4, and 8 hr in culture or with lower PB concentrations. DDT at 10 micrograms/ml decreased cAMP levels in the hepatocytes after 1, 2, 4, and 8 hr of treatment. No effects were seen after 8 hr of treatment or with lower DDT concentrations. DDT (10 micrograms/ml) also decreased cAMP levels in 24-hr-old cultures while PB (500 micrograms/ml) had no effect. Addition of dibutyryl cAMP (0.1 mM) or caffeine (1.0 mM) to freshly plated cultures elevated cAMP levels 50-fold and twofold, respectively. These data suggest that the inhibition of mouse hepatocyte intercellular communication by PB and DDT at the highest concentrations tested may be mediated by transient decreases in intercellular cAMP levels.     

The pharmacokinetics of 1,2-diethyl-3-hydroxypyridin-4-one (CP94) in rats.

The pharmacokinetics of 1,2-diethyl-3-hydroxypyridin-4-one (CP94) and its 2-(1-hydroxyethyl) metabolite (metabolite A) were examined in male Wistar rats using a chronically cannulated conscious-rat model. Serial blood samples were assayed by a reversed phase HPLC method with UV detection. Following iv doses of 25, 50, and 100 mg/kg, the parent compound was eliminated from blood in a biexponential fashion with an average systemic clearance of 1.5 liters/hr/kg. The mean terminal elimination half-life was 2.02 hr and the mean volume of distribution at steady state was 2.69 liters/kg. The areas under the curve (AUCs) for the 25, 50, and 100 mg/kg iv doses were 15, 36, and 72 micrograms/ml/hr, respectively, suggesting that the disposition of CP94 in rats obeys linear kinetics. The oral bioavailability of CP94 (100 mg/kg) was about 53%. Peak blood concentration occurred at about 0.5 hr after oral administration. Following iv doses of CP94 at 25, 50, and 100 mg/kg, metabolite A peaked at about 0.75 hr.     

Hydrolytic and metabolic characteristics of the esters of 1-(3'-hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41), potentially useful iron chelators

1-(3'-Hydroxypropyl)-2-methyl-3-hydroxypyridin-4-one (CP41) has been extensively investigated as an orally effective iron chelator. In order to improve the pharmacokinetic and metabolic properties of CP41, eleven aromatic esters have been synthesised and tested as potential prodrugs. In the present study, the hydrolytic rates of these CP41 esters in phosphate buffer (pH2.0 and pH 7.4), rat blood and rat liver homogenate have been determined and found to cover a wide range. Generally, they possessed relatively slow hydrolytic rates in phosphate buffer (0-50 nmol/ml/hr at pH 2.0 and 0-140 nmol/ml/hr at pH 7.4). The hydrolytic rates in rat blood fell in the range of 9-5766 nmol/ml blood/hr and in rat liver homogenate 1-800 micromol/g liver tissue/hr. All esters possess a higher lipophilicity than that of the parent compound CP41. Although no apparent relationship was observed between the lipophilicities and hydrolytic rates, the esters with relatively higher hydrolytic rates in liver homogenate tend to possess higher iron scavenging efficacies. Further investigation of the metabolism of selected CP41 esters indicates that metabolism is a key factor influencing the efficacy of CP41 esters, as some esters can be metabolically inactivated in the liver in preference to undergoing ester hydrolysis. Ester design, combined with a knowledge of the prodrug metabolism, is a useful strategy for the production of 3-hydroxypyridin-4-ones with enhanced iron scavenging efficacy.     

A model combining the whole embryo culture with human liver S-9 fraction for human teratogenic prediction

The whole embryo culture system has proved particularly useful in evaluating the role of biotransformation in dysmorphogenic processes. Using the traditional method, information on the teratogenic potential of chemicals can be obtained, but the results are difficult to extrapolate to humans. In this study, a human liver S-9 fraction was used as an enzyme source for the bioactivation of cyclophosphamide (CP) in vitro, to assess whether this model mimics more nearly the human situation. CP is a well known rodent teratogen but probably is not teratogenic in humans. The effects were compared of Aroclor-1254-induced rat liver S-9, non-induced rat liver S-9 and human liver S-9 fractions on CP teratogenicity in vitro. CP (30 μg/ml) with non-induced rat S-9 (50 μl) did not cause a significant increase in adverse effects (46.7%), whereas 100% dysmorphogenic effects were shown with induced rat S-9 (30 μl, 50 μl). CP (30–150 μg/ml) did not produce dysmorphogenesis (below 35.5%) in the presence of human S-9 (50 μl).     

Effects of S-21007, a potent 5-HT3 partial agonist, in mouse anxiety.

AIM: To study the effect of S-21007, a 5-HT3 partial agonist in different animal models of anxiety in mice. METHODS: S-21007 effects were evaluated in the behavior tests after intraperitoneal and oral acute treatment or in the light/dark test after both acute and chronic treatments. RESULTS: S-21007 presented anxiolytic-like properties after acute administration in the light/dark box test, the mirrored chamber test, and the elevated plus-maze at low doses 10 ng.kg(-1)-100 micrograms.kg-1, 1-100 micrograms.kg-1 and 10-100 micrograms.kg-1, respectively. In the light/dark box test, S-21007 was active orally after acute treatment at 100 ng.kg(-1)-10 mg.kg-1 and after chronic treatment (14 d) at 1-10 micrograms.kg-1. S-21007 was devoid of sedative or stimulatory effects. CONCLUSION: S-21007 exhibited anxiolytic-like properties. The mechanism of action may be a desensitization of 5-HT3 receptor or an antagonist activity on the 5-HT3 receptors.     

Morphological transformation of BALB/3T3 mouse embryo cells in vitro by vomitoxin

The transforming potential of vomitoxin, a trichothecene mycotoxin produced on cereal grains by fungi of the genus Fusarium, was assessed using mouse embryo BALB/3T3 A31-1-1 cells. Cells grown in Eagle's basal medium with Earle's salts supplemented with 7.5% foetal bovine serum were treated with highly purified vomitoxin, which was dissolved in distilled water and filter-sterilized. Assays were conducted using cells from three different passages at dose levels ranging from 0.1 to 1.6 microgram/ml. The treatment time was 48 hr and the highest dose levels tested produced approximately 10% survival as determined by in situ cell counts. Distilled water and 3-methylcholanthrene (5.0 micrograms/ml) were used as the vehicle and positive controls, respectively. Of the 20 dishes examined per dose group, the numbers of type III foci were 0-1 in the solvent control, 12-15 in the positive control and 0-9 in the treated groups. Comparison of the three assays showed that the level of response varied with passage number. Of the three passages of cells tested-passage numbers 6, 8 and 9 (p6, p8 and p9)--passage-9 cells produced the strongest positive effect.     

Genotoxicity studies of cefmatilen hydrochloride hydrate (S-1090)

Cefmatilen hydrochloride hydrate (S-1090), a new non-ester type of orally active cephem antibiotic synthesized in Shionogi Research Laboratories, was evaluated for its genotoxic potential using three assay systems. In a reverse mutation test with bacteria of Salmonella typhimurium TA100, TA1535, TA98, and TA1537, and Escherichia coli WP2uvrA using the preincubation method, the number of revertant colonies in the S-1090 treated plates was almost equal to that in the negative control plates in all strains with and without metabolic activation system with S9 mix (maximum dose, 100 micrograms/plate in TA98). In a chromosomal aberration test with cultured Chinese hamster lung cells (CHL/IU), S-1090 did not induce structural chromosome aberrations or polyploid cells either in the absence or presence of S9 mix up to the 50% growth inhibition doses. The potential of inducing clastogenicity and/or disruption of mitotic apparatus in vivo by S-1090 was evaluated by a micronucleus test with bone marrow cells of male Jc1:ICR mice. S-1090 suspended in 0.5% aqueous methylcellulose was administered by oral gavage up to 2000 mg/kg/day in single and double dosing groups. No induction of micronucleated polychromatic erythrocytes was observed 24 hr after the last dosing in each group. As all three genotoxicity tests showed negative responses, S-1090 is thought to have no genotoxic potential.     

Effect of nerve growth factor and thyrotropin releasing hormone on cholinergic neurones in developing rat brain reaggregate cultures lesioned with ethylcholine mustard aziridinium

Foetal rat whole brain reaggregate cultures were prepared in a serum-supplemented (S+) or serum-free medium (S-). Ethylcholine mustard aziridinium (ECMA) was added to the cultures at 9 days in vitro (DIV) at concentrations of 12.5, 25 or 50 microM. Choline acetyltransferase (ChAT) activity was measured at +2, +48 and +96 hr following treatment. In certain experiments the neurotrophic factors, thyrotropin releasing hormone (TRH: 50 micrograms/ml, daily from 9 DIV) or nerve growth factor (NGF: 7S subunit, 5 ng/ml, 0 and +48 hr following ECMA) were added during ECMA treatment. In both types of reaggregate cultured in S+ and S- media there was a 40-80% loss of ChAT activity following ECMA exposure (final concentration = 12.5 microM), presumed to reflect cholinergic cell loss. In both S+ and S- brain reaggregates NGF produced increased ChAT activity with more marked effects in S+ (45-55% increase, +48-96 hr) than in S- medium (20-25% increase, 2-96 hr). No effect on cholinergic muscarinic receptors (specific 3H-QNB binding) was evident after treatment with NGF. TRH had no effect on ChAT activity in the S+ cultures but produced small increases in the S- culture condition (approx 20%, +2-48 hr). Despite a residual "ECMA-resistant" pool of ChAT in the cultures, neither neurotrophic agent was found to cause a reversal of the lesion. In conclusion, the cholinotoxin ECMA appears to produce a cholinergic deficit in both developing S+ and S- reaggregates. This was not reversible by NGF or TRH at the concentrations and under the conditions tested. NGF had marked effects on ChAT activity without affecting muscarinic receptors in untreated developing brain reaggregates cultured in an S+ medium.     

The metabolic chiral inversion of 2-phenylpropionic acid in rat, mouse and rabbit

The metabolic chiral inversion of the 2-arylpropionic acids has been investigated in laboratory animals, using the simplest congener, 2-phenylpropionic acid, as a model compound. The chiral inversion was found to occur after administration of the racemate to the rat and rabbit, but not in the mouse. The formation of the ester glucuronide was enantioselective for the S-(-)-isomer in the rat and mouse, but showed no stereoselectivity in the rabbit. [corrected] In the rat, the extent of inversion from R-(-) to S-(+) was greater at a dose of 30 mg/kg than at 150 or 300 mg/kg. The enantiomeric composition of the acid in urine was the same when the racemate was given orally or by i.p. injection. When the R-(-)isomer was given to rats, some 30% of the excreted acid was in the S-(+)-form, but when the S-(+)-isomer was given, the inversion was much less evident. In this case, the S/R ratio of the excreted phenylproprionic acid was ca 9:1. Following the administration of the racemate to rats, the plasma elimination half-life of the R-(-)-form was shorter (3.0 vs 4.8 hr for the S-(-)-isomer); this was due to its considerably greater plasma clearance (65.9 vs 43.6 micrograms/ml hr), since the volumes of distribution of the enantiomers were the same. The S/R ratio of 2-phenylpropionic acid in plasma rose progressively with time, from 1:1 in the dose solution to 2.1:1 at 8 hr.     

Hydrolytic and Metabolic Characteristics of the Esters of1‐(3′‐Hydroxypropyl)‐2‐methyl‐3‐hydroxypyridin‐4‐one (CP41), Potentially Useful Iron Chelators

1‐(3′‐Hydroxypropyl)‐2‐methyl‐3‐hydroxypyridin‐4‐one (CP41) has been extensively investigated as an orally effective iron chelator. In order to improve the pharmacokinetic and metabolic properties of CP41, eleven aromatic esters have been synthesised and tested as potential prodrugs. In the present study, the hydrolytic rates of these CP41 esters in phosphate buffer (pH2.0 and pH7.4), rat blood and rat liver homogenate have been determined and found to cover a wide range. Generally, they possessed relatively slow hydrolytic rates in phosphate buffer (0–50 nmol/ml/hr at pH 2.0 and 0–140 nmol/ml/hr at pH 7.4). The hydrolytic rates in rat blood fell in the range of 9–5766 nmol/ml blood/hr and in rat liver homogenate 1–800 μmol/g liver tissue/hr. All esters possess a higher lipophilicity than that of the parent compound CP41. Although no apparent relationship was observed between the lipophilicities and hydrolytic rates, the esters with relatively higher hydrolytic rates in liver homogenate tend to possess higher iron scavenging efficacies. Further investigation of the metabolism of selected CP41 esters indicates that metabolism is a key factor influencing the efficacy of CP41 esters, as some esters can be metabolically inactivated in the liver in preference to undergoing ester hydrolysis. Ester design, combined with a knowledge of the prodrug metabolism, is a useful strategy for the production of 3‐hydroxypyridin‐4‐ones with enhanced iron scavenging efficacy.     

Genotoxicity studies of stevia extract and steviol by the comet assay

The genotoxicity of steviol, a metabolite of stevia extract, was evaluated for its genotoxic potential using the comet assay. In an in vitro study, steviol at 62.5, 125, 250, and 500 micrograms/ml did not damage the nuclear DNA of TK6 and WTK1 cells in the presence and absence of S9 mix. In vivo studies of steviol were conducted by two independent organizations. Mice were sacrificed 3 and 24 hr after one oral administration of steviol at 250, 500, 1000, and 2000 mg/kg. DNA damage in multiple mouse organs was measured by the comet assay as modified by us. After oral treatment, stomach, colon, liver, kidney and testis DNA were not damaged. The in vivo genotoxicity of stevia extract was also evaluated for its genotoxic potential using the comet assay. Mice were sacrificed 3 and 24 hr after oral administration of stevia extract at 250, 500, 1000, and 2000 mg/kg. Stomach, colon and liver DNA were not damaged. As all studies showed negative responses, stevia extract and steviol are concluded to not have DNA-damaging activity in cultured cells and mouse organs.     

Effects of nisoldipine on recovery of coronary blood flow, sarcoplasmic reticulum function and other biochemical parameters in post-ischaemic porcine myocardium

The effects of nisoldipine (0.1 micrograms/kg/min; n = 9) or its solvent (n = 9) were studied in pigs, in which left anterior descending coronary artery (LADCA) blood flow in both groups was reduced to 20% of baseline for 60 min and reperfused for 2 hr. Infusions were started at 30 min of ischaemia and lasted throughout reperfusion. In both groups, flow reduction abolished regional contractile function and caused similar decreases in the level of creatine phosphate (CP; by 70%) and the energy charge (from 0.91 to 0.69), mean arterial blood pressure (by 25%), LVdP/dtmax (by 30%) and cardiac output (by 30%). During ischaemia LADCA blood flow slightly increased (from 14 +/- 8 to 24 +/- 6 mL/min/100 g; P less than 0.05) in the nisoldipine-treated animals, resulting in an increase in CP to 91 +/- 24% of baseline and preventing further decreases in energy charge, as observed in the solvent-treated animals. After 2 hr of reperfusion in neither group return of contractile function of the post-ischaemic myocardium was observed. Post-ischaemic blood flow in the nisoldipine-treated pigs increased from 24 +/- 6 mL/min/100 g to 76 +/- 14 mL/min/100 g and from 19 +/- 6 mL/min/100 g to 41 +/- 6 ml/min/100 g in the solvent-treated animals (P less than 0.05) after 2 hr of reperfusion. Myocardial work was significantly higher in the nisoldipine-treated animals (111 +/- 15 mmHg.L/min as compared to 69 +/- 14 mmHg.L/min in the solvent-treated pigs after 2 hr of ischaemia). The energy charge of the post-ischaemic myocardium was similar for both groups (0.84 +/- 0.02 for the nisoldipine-treated and 0.83 +/- 0.03 for the solvent-treated animals). The rate of sarcoplasmic reticular Ca2+ uptake of the non-ischaemic segment of the nisoldipine-treated animals was 61% higher (P less than 0.05) than that of the solvent-treated animals. In the post-ischaemic myocardium similar rates of Ca2+ uptake were found in both groups, but the activities were markedly lower as compared to the non-ischaemic myocardium. It is concluded that nisoldipine increases blood flow during reperfusion, which may have been caused by coronary vasodilatation. However, attenuation of the "no-reflow" phenomenon also contributed, since more rapid rephosphorylation of ADP leading to an increase in CP during ischaemia may have preserved jeopardized cells. Moreover, nisoldipine increases the sarcoplasmic reticular Ca2+ pump activity independent of ischaemia, which may have contributed in reducing the Ca2+ overload.     

Effect of growth hormone on caffeine metabolism in hypophysectomized rats

Two groups of six male Sprague-Dawley hypophysectomized rats (operated on day 0), 8 weeks old, treated by sc tetracosactid (ACTH, 10 micrograms every 24 hr), thyroxine (5 micrograms/100 g every 24 hr) and desmopressin (240 ng/kg/24 hr continuous infusion) received SC either saline (group I) or human growth hormone (hGH, 120 micrograms/24 hr) (group II) continuous infusion. ACTH and thyroxine were administered on days 7-19 and desmopressin and hGH on days 8-19, after surgery. They received po caffeine 4 mg/kg as citrate salt on day 15. The 0-12, 12-24 and 24-48 hr urine samples were collected after caffeine administration. Caffeine and metabolites concentrations in urines were determined using HPLC. Effect on hGH on caffeine metabolism was assessed comparing group I and group II. In 0-48-hr urine, 1-methylxanthine (154 +/- 169 pmol/g) and 3-7-dimethyluric acid (5.57 +/- 19.3 pmol/g) in group II were significantly lower than in group I (391 +/- 340 pmol/g and 262 +/- 338 nmol/g, respectively) (p less than 0.05). Other metabolites (6-amino-5-(N-methyl formylamino)-1,3-dimethyluracil included) excretion was not altered. Total, N3-, N7- and N1-demethylation ratios on 0-48 hr urine were not modified by hGH treatment. However, demethylation ratios on 12-24 and 24-48 hr (N3 + N7 + N1) and on 24-48 hr urine samples (N3 and N7) were significantly reduced in group II (p less than 0.05) suggesting an increase in the rate of appearance of demethylated metabolites during hGH treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of KT-362, a new Na and Ca influx and Ca release inhibitor, on canine ventricular arrhythmias

Antiarrhythmic effects of the new drug KT-362, which was reported to suppress Na and Ca currents of cardiac cells and also to suppress intracellular Ca release in isolated smooth muscle preparations, were examined using two-stage coronary ligation-, digitalis- and adrenaline-induced ventricular arrhythmias in the dog. Intravenous KT-362 at 10 mg/kg suppressed coronary ligation arrhythmia both at 24 and 48 hr after ligation, and the minimum effective plasma concentrations for arrhythmias induced by 24 hr coronary ligation and 48 hr coronary ligation were 6.1 +/- 1.7 and 8.6 +/- 2.7 micrograms/ml, respectively. Antiarrhythmic effects were accompanied by transient hypotension. Oral administration of 70-100 mg/kg was also effective on 24 hr coronary ligation arrhythmia. However, there was no prominent hypotension in these experiments. Intravenous KT-362 at 3 mg/kg suppressed digitalis arrhythmia; and the minimum effective plasma concentration was 3.3 +/- 1.2 micrograms/ml, which was lower than the effective plasma concentrations for coronary ligation arrhythmias. Intravenous KT-362 at 1 mg/kg also suppressed adrenaline arrhythmia; and the minimum effective plasma concentration was 1.0 +/- 0.1 microgram/ml, the lowest among the effective plasma concentrations. These pharmacological profiles of KT-362 are quite different from those of class 4 Ca antagonists, but similar to those of class 1 drugs such as propafenone. Though KT-362 has a hypotensive effect, it is effective on canine ventricular arrhythmias; thus its clinical usefulness for supraventricular and ventricular arrhythmias is expected.     

Platelet-derived growth factor (PDGF) accelerates induction of competence, and heparin does not inhibit PDGF-induced competence in primary cultured smooth muscle cells of rat aorta.

The time-dependent effect of platelet-derived growth factor (PDGF) on cell proliferation was investigated to clarify whether PDGF accelerates the rate of proliferation or its start in primary cultured smooth muscle cells (SMC) of rat aorta. In synchronized SMC at the G0 phase, 1, 10 and 100 ng/ml PDGF started DNA synthesis at 24, 15-18 and 12 hr, respectively, after stimulation by 3% fetal bovine serum (FBS) or hypophysectomized rat plasma (deficient in insulin-like growth factor-I (IGF-I)). Heparin (1, 10 or 100 micrograms/ml) decreased only the rate of DNA synthesis stimulated by PDGF in synchronized SMC. DNA synthesis in non-synchronized cells stimulated by PDGF with FBS was determined up to 10 days in culture. The stimulation with 1% FBS plus 30 ng/ml PDGF potentiated the DNA synthesis which was saturated with stimulation by 10% FBS alone, suggesting that prolonged treatment of PDGF transforms SMC. These results demonstrated that PDGF concentration-dependently accelerated the induction of competence independently of IGF-I, and heparin did not inhibit PDGF-induced competence but inhibited progression in primary cultured SMC of rat aorta.     

Cytotoxic effects of amiodarone and desethylamiodarone on human thyrocytes

Since recent in vivo evidence suggests that the benzofuran antiarrhythmic drug amiodarone has a direct toxic effect on the human thyroid gland, we have investigated the effects of both amiodarone and its metabolite desethylamiodarone on a novel immortalized functional human thyrocyte line (SGHTL-34 cells). Desethylamiodarone markedly reduced cell number as assessed from both DNA and protein content. Few cells were left after 24 hr exposure to 12.5 micrograms/ml; the concentration producing death of 50% of cells (EC50) was 6.8 +/- 1.1 micrograms/ml (mean +/- SE, N = 15). Amiodarone was much less potent, producing a maximum decrease in cell number of approximately 25% at concentrations up to 50 micrograms/ml. The effect of desethylamiodarone was seen within 24 hr of culture. T3 in concentrations up to 0.75 micrograms/ml had no effect on the action of amiodarone or desethylamiodarone on SGHTL-34 cells. Light microscopy demonstrated vacuolation of SGHTL-34 cells after 4-day culture with either the drug or its metabolite. Studies using primary cultures of human retroorbital fibroblasts demonstrated that the greater cytotoxicity of desethylamiodarone was not confined to thyrocytes. When SGHTL-34 cells were incubated with 2.5 micrograms/ml desethylamiodarone for 4 days, 71.7 +/- 0.9% was taken up by the cells; there was no detectable conversion to amiodarone. Incubation of thyrocytes with 50 micrograms/ml amiodarone for 4 days resulted in the uptake of 80.1 +/- 2.1% by the cells. In addition, 5.0 +/- 0.1% of the amiodarone was converted to material with the same retention time as desethylamiodarone standard; of this material, 72.9 +/- 2.8% was taken up by the cells. We conclude that desethylamiodarone, at concentrations near those found in the plasma of patients on long-term amiodarone therapy, exerts a direct cytotoxic effect on human thyroid cells in short-term culture. This effect may play a role in the aetiology of clinical thyroid disease during amiodarone therapy. We suggest that, since the effect is not restricted to thyrocytes, desethylamiodarone may play a role in the aetiology of amiodarone toxicity which occurs clinically in many tissues.     

In vitro embryotoxicity and teratogenic potential of etretinate and etretin

The embryotoxicity and teratogenic potential of etretinate and its main metabolite, etretin, with or without enzymatic drug activation, were investigated in vitro using the rat whole embryo culture system. The metabolizing system used was either a rat liver homogenate (S-9 mix) or esterase (carboxylic-ester hydrolase). Groups of 8–14 rat embryos (explanted on day 9.5 of pregnancy) were cultured for 48 hr with 0, 3, 10, 30 or 100 μg etretinate/ml with or without S-9 mix, or with 0, 0.03, 0.1, 0.3, 1, 3 or 10 μg etretinate/ml in the presence of 0.5 or 1 U esterase/culture flask. Yolk-sac diameters and vascularization and crown-rump and head lengths were unaffected by etretinate treatment alone but somite numbers and morphological scores were reduced at 30 and 100 μg etretinate/ml. The number of embryos with morphological anomalies was increased in the presence of 10 μg etretinate/ml but not significantly so. At higher concentrations the incidence of embryos with anomalies was 100%. Activation of etretinate in the presence of S-9 mix led to increased teratogenic activity of the test compound in vitro, although embryonic crown-rump and head lengths were comparable to control measurements up to the highest concentrations tested. When it was co-incubated with esterase, etretinate became 100 times more active, and its effects occurred in the same concentration range as the embryotoxic effects of etretin added to the culture directly. Without activation etretinate was approximately 100 times less active than etretin in producing general embryotoxicity as well as anomalies in the in vitro culture. The anomalies found after in vitro exposure of rat embryos to etretinate or etretin in this study resembled to a great extent the malformations found in vivo in several species including man. The pattern of anomalies was specific for the compounds used and was similar to that induced by other retinoids. The concentrations of etretin eliciting teratogenic effects in vitro were in the range of etretin peak plasma levels during etretinate therapy in man. Our results indicate that the whole-embryo culture system is a sensitive and valuable model for assessing the embryotoxic and teratogenic potential of retinoids in vitro.