N-vinylpyrrolidone dimer (VPD), a novel excipient for oral drugs: repeat-dose oral toxicity in Sprague-Dawley rats.

N-vinylpyrrolidone dimer (VPD), a novel vehicle for preclinical toxicity studies, was evaluated in a standard 28-day oral toxicity study in rats including a 4 week recovery period. In addition, a subgroup of animals was dosed for 13 weeks. In the 28-day study arm, daily dosages of 0 (saline control, 3mL/kg), 300, 1000 or 3000mg/kg at respective dose volumes of 0.3, 1 and 3mL/kg were administered. In the 13 week study arm, animals received daily doses of 0 or 300mg/kg. No test item related mortality or changes in body weight, food consumption, ophthalmology and clinical pathology parameters were observed after 28-days or 13 weeks of administration. VPD induced transient salivation at all tested dose levels after each dose and increased water consumption at doses ≥1000mg/kg (28-day arm). After 28-days of administration, urinalysis revealed slightly higher mean specific gravity in all treated groups. Relevant organ weight changes consisted of increased mean liver weights. Histopathology revealed hepatocellular centrilobular hypertrophy at a dose-related incidence and severity, minimal to slight follicular cell hypertrophy in male thyroids, hypertrophy of basophil/chromophobe cells in pituitaries and an increase in kidney hyaline droplets consistent with α(2µ)-globuline immunohistochemistry. After a 4-week recovery, all changes were partially or completely reversible. Administration of VPD at 300mg/kg for 13 weeks caused similar histopathological findings observed after 28-days dosing. Overall, in rats, repeated high oral doses of VPD produced changes in the liver, thyroid and pituitary, most likely secondary to hepatic microsomal enzyme induction and stimulation of the thyroid via disruption of the hypothalamic-pituitary-thyroid axis. In conclusion, our data suggest that VPD is a promising vehicle for preclinical studies. However, in rats, findings secondary to hepatic microsomal enzyme induction and stimulation of the thyroid need to be taken into consideration, depending on dose and study duration. In order to develop VPD as a preclinical excipient, additional preclinical toxicity studies (non-rodents, different administration routes, chronic and reproductive toxicity) would be beneficial.

Comparison of haematology, coagulation and clinical chemistry parameters in blood samples from the sublingual vein and vena cava in Sprague-Dawley rats.

The investigation of clinical pathology parameters (haematology, clinical chemistry and coagulation) is an important part of the preclinical evaluation of drug safety. However, the blood sampling method employed should avoid or minimize stress and injury in laboratory animals. In the present study, we compared the clinical pathology results from blood samples collected terminally from the vena cava (VC) immediately before necropsy with samples taken from the sublingual vein (VS) also prior to necropsy in order to determine whether the sampling method has an influence on clinical pathology parameters. Forty-six 12-week-old male Sprague-Dawley rats were assigned to two groups (VC or VS; n = 23 each). All rats were anaesthetized with isoflurane prior to sampling. In the VC group, blood was withdrawn from the inferior VC. For VS sampling, the tongue was gently pulled out and the VS was punctured. The haematology, coagulation and clinical chemistry parameters were compared. Equivalence was established for 13 parameters, such as mean corpuscular volume, white blood cells and calcium. No equivalence was found for the remaining 26 parameters, although they were considered to be similar when compared with the historical data and normal ranges. The most conspicuous finding was that activated prothrombin time was 30.3% less in blood taken from the VC (16.6 ± 0.89 s) than that in the VS samples (23.8 ± 1.58 s). Summing up, blood sampling from the inferior VC prior to necropsy appears to be a suitable and reliable method for terminal blood sampling that reduces stress and injury to laboratory rats in preclinical drug safety studies.

Role of metabolic activation in the carcinogenicity of estrogens: studies in an animal liver tumor model.

Male Syrian golden hamsters chronically exposed to certain synthetic estrogens such as diethylstilbestrol (DES) or 17 alpha-ethinylestradiol (EE2) and fed a diet containing 7,8-benzoflavone (BF) develop a high incidence of liver tumors. No such tumors are found in animals treated with estrogen or BF alone. To clarify the role of metabolic activation of the estrogen and BF in the mechanism of hepatocarcinogenesis in this animal model, the effects of pretreatment with DES and EE2 alone and in combination with BF on the metabolism of DES, EE2, and BF in hepatic microsomes, isolated hepatocytes, and hamster bile were studied. Hamsters were pretreated for up to 32 weeks. The results clearly show that DES metabolism was not significantly modified under any pretreatment regimen. EE2 metabolism exhibited a slight increase in 2-hydroxylation after pretreatment with BF and with BF plus EE2. The most pronounced effect was observed in BF metabolism after pretreatment with BF, with BF plus DES, and with BF plus EE2: the metabolic rate was increased and several new metabolites that were not found in untreated or estrogen-pretreated animals were formed. These metabolites were tentatively identified as BF-dihydrodiol and dihydroxy-BFs. The formation of these new BF metabolites was accompanied by a change in the activities of certain cytochrome P-450 isoenzymes in hamster liver microsomes. The results of this study imply that metabolic activation of BF rather than of the estrogens plays an important role in the mechanism of carcinogenesis in this animal liver tumor model.

Effects of various inducers on diethylstilbestrol metabolism, drug-metabolizing enzyme activities and the aromatic hydrocarbon (Ah) receptor in male Syrian golden hamster liver.

In order to elucidate the role of metabolic activation of the synthetic estrogen, diethylstilbestrol (DES), in the mechanism of liver tumor formation in male Syrian golden hamsters observed after combined treatment with DES and 7,8-benzoflavone (7,8-BF), the metabolism of DES and the concentrations and activities of various drug-metabolizing enzymes were studied in hamster liver microsomes after various pretreatments. The levels of the hepatic aromatic hydrocarbon (Ah) receptor were also determined. Pretreatment with 7,8-BF increased both P450 and cytochrome b5 levels, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induced P450 but not cytochrome b5. 7,8-BF pretreatment increased 7-ethoxyresorufin-O-deethylase (EROD) 3-fold and 7-pentoxyresorufin-O-dealkylase (PROD) 2.5-fold, whereas aromatic hydrocarbon hydroxylase (AHH) and 7-ethoxycoumarin-O-deethylase (ECOD) activities were only slightly induced by 7,8-BF. MC pretreatment increased EROD 8-fold and PROD activity 7-fold, whereas PB pretreatment enhanced AHH 4.5-fold and PROD activity 4-fold. In contrast to PB, pretreatment with 7,8-BF and MC reduced the oxidative metabolism of DES in hepatic microsomes, but the pattern of metabolites was identical with that in untreated controls. Treatment of hamsters with the inducers changed the hepatic Ah receptor level. PB and MC-pretreatment resulted in an increase of the receptor level 1.5-fold and 1.3-fold, respectively, whereas 7,8-BF-pretreatment leads to a 1.5-fold decrease. The dissociation constant Kd is 170 nM for the reaction of 7,8-BF with the hamster Ah receptor compared to 70 nM for 5,6-BF and 38 nM for 2,3,7,8-tetrachlorodibenzofuran (TCDF). The Kd-value is 3.6 nM for TCDF with the rat receptor protein. It is concluded from these data that metabolic activation of DES is not involved in the mechanism of hepatocarcinogenesis in this animal tumor model.

Modification of 7,8-benzoflavone metabolism in hamster liver and kidney microsomes by hepatic tumor inducing treatments.

The effect of pre-treatment of male Syrian golden hamsters with 7,8-benzoflavone (BF), with diethylstilbestrol (DES) and with BF plus DES on the metabolism of [14C]BF in hepatic and renal microsomes has been studied in vitro. Whereas hepatic microsomes from DES-treated animals produced the same pattern of BF metabolites as control microsomes, a marked quantitative and qualitative alteration of BF metabolism was observed with liver microsomes from animals pre-treated with BF and with BF plus DES: the metabolic rate was increased and three new metabolites were formed which were not observed with control hepatic microsomes. These metabolites, which were tentatively identified as BF-5,6-dihydrodiol and two isomeric dihydroxy-BFs, were not detected in incubations with renal microsomes under any pre-treatment regimen. Non-extractable binding of radioactivity to hepatic and renal microsomal protein was observed in all incubations but did not exhibit as pronounced a dependence on pre-treatment as did the pattern of BF metabolites. Based on the metabolic data it is concluded that BF induces its own oxidative metabolism. Among the metabolites are reactive intermediates that bind to cellular macromolecules and may play an important role in tumor formation in the male Syrian hamster liver following prolonged treatment with BF plus DES.

Multiple pathways for the oxidative metabolism of estrogens in Syrian hamster and rabbit kidney.

Microsomal preparations from hamster kidney, a target tissue for the carcinogenic action of stilbene-type and steroidal estrogens, catalyze the oxidative metabolism of diethylstilbestrol (DES). The formation of the major metabolite Z,Z-dienestrol and of reactive intermediates capable of protein binding were mediated by enzyme activities requiring nicotinamide-adenine dinucleotide phosphate (reduced form-NADPH), cumene hydroperoxide, or arachidonic acid (ARA). In addition, hydroxylated DES metabolites were detected in NADPH-supplemented incubations. The NADPH-dependent oxidation of DES was inhibited by SKF 525A and metyrapone. Monooxygenase-catalyzed metabolism was apparently responsible for the majority of DES oxidation in microsomes from whole hamster kidneys in vitro and this activity is preferentially localized in the kidney cortex. However, ARA-dependent, i.e., prostaglandin H synthase (PHS) mediated oxidation of DES and of the catechol estrogen 2-hydroxyestrone was demonstrated as well in the medulla of both rabbit and hamster kidney. It is proposed that monooxygenase and PHS activities act in concert in the metabolic activation of carcinogenic estrogens. This appears to apply in particular to steroidal estrogens, since catechol estrogens formed by monooxygenases are further oxidized to reactive intermediates by PHS and other peroxidatic enzymes.

Effect of pretreatment of male Syrian golden hamsters with 7,8-benzoflavone and with diethylstilbestrol on P-450 isoenzyme activities and on microsomal diethylstilbestrol metabolism.

Combined treatment of male Syrian golden hamsters with the synthetic estrogen diethylstilbestrol (DES) and 7,8-benzoflavone (7,8-BF) gives rise to a high incidence of hepatocellular carcinomas, whereas no such tumors are formed with DES alone nor with 7,8-BF alone. To determine whether alterations in DES metabolism may account for the observed hepatocarcinogenicity, we have studied the effect of pretreatment with 7,8-BF alone, DES alone and 7,8-BF plus DES on the levels of hepatic P-450 and cytochrome b5, on the activities of various P-450 isoenzymes and on microsomal DES metabolism. Hepatic P-450 content was significantly increased after pretreatment with 7,8-BF and decreased after DES, while combined pretreatment led to levels similar to those in untreated control animals. Hepatic cytochrome b5 was also elevated in 7,8-BF-treated hamsters; DES pretreatment had no effect, and combined pretreatment led to a slight increase. Four different substrates were used to probe P-450 isoenzyme activity. Aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin-O-deethylase (ECOD), 7-ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) were all elevated after 7,8-BF-pretreatment, while DES led to a decrease in these activities with the exception of AHH, where a transient increase which was observed after 8 and 20 weeks of pretreatment was back to control levels after 32 weeks. Combined pretreatment with 7,8-BF and DES led to an intermediate response (slight increase) with AHH, EROD and PROD, but not with ECOD, where a full induction comparable with that observed after 7,8-BF alone was elicited. In spite of the modulation of enzyme levels and activities observed after the various pretreatments, the metabolism of DES in microsomes from pretreated animals was virtually identical with that from controls. Therefore it is concluded that modulation of hepatic DES metabolism is not the reason for the observed hepatotumorigenicity; instead, it is speculated that 7,8-BF is the carcinogenic agent in this tumor model, and DES may act as a promotor.

Effect of pretreatment with 7,8-benzoflavone and diethylstilbestrol on the hepatic metabolism of diethylstilbestrol in the male Syrian golden hamster in vivo.

Liver tumors are induced in male Syrian golden hamsters by the combined treatment with diethylstilbestrol (DES) and 7,8-benzoflavone (7,8-BF), but not with either substance alone. With the aim of clarifying whether metabolic activation of DES is involved in the mechanism of tumorigenesis in this animal model, we have studied the effect of pretreatment with 7,8-BF alone, DES alone, and 7,8-BF plus DES for 2, 8, 20 and 32 weeks on the hepatic in vivo metabolism of DES, using biliary metabolites collected from bile-duct cannulated male hamsters as probe. Formation of glucuronides and sulfates was not affected by treatment with 7,8-BF nor 7,8-BF plus DES. In contrast, animals pretreated with DES alone had a decreased amount of glucuronides and an increased proportion of unconjugated material in the bile. Oxidative metabolism of DES was not significantly altered in hamsters treated with 7,8-BF for up to 20 weeks, whereas pretreatment with DES alone and with 7,8-BF plus DES caused an enhancement of oxidative DES metabolism in vivo, leading mostly to highly polar, as yet unidentified products. From a consideration of various cytochrome P-450-associated enzyme activities, it is concluded that the observed effect on biliary DES metabolites is most likely to be due to an estrogen-induced intrahepatic cholestasis. Taken together, the data do not support a role for the metabolic activation of DES in this tumor model. Alternative mechanisms are proposed.

The effects of pretreatment with 7,8-benzoflavone on drug-metabolizing enzymes and diethylstilboestrol metabolism in male hamster liver microsomal preparations.

1. Pretreatment of male Syrian golden hamsters with 7,8-benzoflavone (7,8-BF, 0.4% in the diet) for 4 and 20 weeks resulted in a significant increase in the amount of cytochrome P-450 and cytochrome b5 in hepatic microsomes. The activities of microsomal 7-ethoxycoumarin-O-deethylase (ECOD) and 7-ethoxyresorufin-O-deethylase (EROD) increased by a factor of 2 and 5, respectively, whereas aryl hydrocarbon hydroxylase (AHH) activity was only marginally enhanced. 2. Because the relative increase in cytochrome b5 exceeded that of cytochrome P-450, the ratio of P-450 to b5 decreased from 2.2 in controls to 1.6 in 7,8-BF-treated animals. 3. Hepatic microsomes from untreated and 7,8-BF-pretreated hamsters metabolize E-diethylstilboestrol (E-DES) to its stereoisomer Z-DES and to several oxidative metabolites, of which 3-hydroxy-DES and Z,Z-dienestrol were unambiguously identified by g.l.c.-mass spectrometry. 4. Pretreatment with 7,8-BF led to a marked decrease in the formation of all oxidative DES metabolites but not in the isomerization to Z-DES. The possible implication of these data for the mechanism of liver tumour formation by the combined treatment of hamsters with 7,8-BF and DES is discussed.

Induction of P-450 isoenzyme activities in Syrian golden hamster liver compared to rat liver as probed by the rate of 7-alkoxyresorufin-O-dealkylation.

The activities of 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-deethylase (PROD), 7-ethoxycoumarin-O-deethylase (ECOD) and aromatic hydrocarbon hydroxylase (AHH) were measured in hepatic microsomes from male and female Wistar rats and Syrian golden hamsters in order to probe the basal activity and the inducibility by phenobarbital (PB) and 3-methylcholanthrene (MC) of different P-450 isoenzymes. The basal activities of EROD and ECOD, but not PROD and AHH, were higher in male hamsters than in male rats. No sex-related difference in enzyme activities was observed with hamsters, whereas male rats had a higher ECOD and AHH activity than female rats. Induction by PB led to a 450-fold and 250-fold increase in PROD activity in male and female rat liver microsomes, respectively, while MC had a more pronounced inductive effect on EROD activity in this species. In hamsters, EROD activity was induced by MC but not by PB. Unexpectedly PROD activity in male and female hamster liver microsomes was only moderately induced by PB, the extent being lower than on induction by MC. Therefore, the activity of PROD, which is useful as a specific enzymatic assay for P-450 IIB in the rat liver, cannot be used to probe PB-like inducers in the hamster liver.

Metabolism of diethylstilbestrol in hamster hepatocytes.

Under certain modulating conditions the liver of the male Syrian golden hamster is a target organ for the carcinogenic effect of the synthetic estrogen diethylstilbestrol (DES). As a basis for mechanistic studies aimed at elucidating the role of metabolic activation in the process of DES-induced neoplasia, the metabolism of 14C-DES was investigated in freshly isolated hamster hepatocytes. These oxidative metabolites of DES, viz. Z,Z-dienestrol,3'-hydroxy-DES and 1-hydroxy-E-DES, were formed in 14.2, 9.1, and 0.3% yield, respectively, when hepatocytes were incubated with 50 nmol DES/mg cellular protein for 60 min. Glucuronides (4.0%) and sulfates (2.8%) of DES and of the oxidative metabolites were also found, and non-extractable binding of radioactivity to cellular protein was observed indicating the formation of reactive intermediates. The capability of hamster hepatocytes to oxidize and conjugate DES should allow the investigation of the effects of modulators on the metabolic activation of DES in this cellular system in order to help clarify the mechanisms of DES-induced hepatocarcinogenesis.

Effect of 7,8-benzoflavone pretreatment on diethylstilbestrol metabolism, drug-metabolising enzymes and the aromatic hydrocarbon (Ah) receptor in male hamster liver.

Pretreatment of male Syrian golden hamsters with 7,8-benzoflavone (7,8-BF) leads to a marked increase of cytochrome P450 and cytochrome b5 levels in the liver, whereas phenobarbital (PB) and 3-methylcholanthrene (MC) induce cytochrome P450 but not cytochrome b5 7,8-BF pretreatment has only minor effects on the activities of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin-O-deethylase, but 7-ethoxyresorufin-O-deethylase is increased 3-fold. In contrast to PB, pretreatment with 7,8-BF or MC reduces the oxidative metabolism of diethylstilbestrol (DES) by hepatic microsomes in vitro. The cytosolic level of the aromatic hydrocarbon (Ah) receptor in hamster liver is decreased by 7,8-BF and slightly enhanced by MC pretreatment. PB increases the receptor level 1.5-fold. The affinity of 7,8-BF to the Ah receptor in vitro is of the same order of magnitude as that of the known ligands 5,6-benzoflavone and 2,3,7,8-tetrachlorodibenzofurane. PB and DES show no binding to the receptor protein.

Reference values for free amino acids and other biochemical constituents in serum of male rabbits.

Reference values for free amino acids in male rabbits (n = 145) were determined by gas chromatography. In addition, reference values for glucose, serum transaminase activities (aspartate aminotransferase, alanine aminotransferase), and serum protein fractions are reported.

On the state of calcium ions in isolated rat liver mitochondria. V. Development of a rapidly dischargeable pool of mitochondrial calcium during calcium-induced transition.

Ruthenium red-induced calcium efflux from rat liver mitochondria is accelerated in the course of calcium-mediated mitochondrial transition. Analysis of the efflux patterns reveals biexponential kinetics consisting of the native slow phase preceded by a faster phase developing in the presence of calcium. The data are consistent with a progressive transformation of mitochondrial calcium into a rapidly dischargeable pool prior to spontaneous calcium release. Analysis of the efflux pattern is proposed as a method to discriminate between different mechanisms of modulation of ruthenium red-induced calcium efflux. Thus, it is shown that acetate, in contrast to phosphate, stimulates ruthenium red-induced calcium efflux due to the development of the rapidly dischargeable pool of calcium.

On the state of calcium ions in isolated rat liver mitochondria IV. Prevention of phosphate-induced mitochondrial destruction by ruthenium red-insensitive calcium release.

Ruthenium red prevented the spontaneous calcium release and the accompanying mitochondrial destruction occurring in calcium-loaded mitochondria in the presence of phosphate. Under these conditions delta pH and membrane potential delta psi were preserved and the ruthenium red-induced calcium efflux was low and at a constant rate. On prolonged incubation with calcium prior to addition of ruthenium red increasingly more mitochondrial calcium developed into a pool rapidly dischargeable by ruthenium red. This development was accompanied by stimulation of respiration which was, however, not abolished by ruthenium red as could have been expected if it had been caused by calcium cycling. Calcium therefore altered mitochondria by a different mechanism than by cycling across the inner membrane.

On the state of calcium ions in isolated rat liver mitochondria. III. Diversity of ruthenium red action on different calcium pools.

Calcium efflux from isolated mitochondria on ruthenium red addition was shown to be biphasic. The rate of efflux from a slowly releasable pool was independent of preincubation. It could be saturated and in extrapolation revealed a maximal rate of 3.6 nmol/(min X mg protein). The efflux from a second, rapidly dischargeable pool was related to calcium added up to 300 nmol/mg protein when a final rate of 15 nmol/(min X mg protein) was reached. The magnitude of the latter pool depended on the time of preincubation in the presence of calcium and correlated with mitochondrial swelling. After ruthenium red addition, a further increase of this pool and spontaneous, destructive calcium release was prevented. Three conclusions are drawn from these results: On preincubation with calcium, part of the mitochondrial calcium develops into a rapidly dischargeable pool. This pool is responsible for mitochondrial alterations resulting in a spontaneous, destructive release of total calcium. Ruthenium red inhibits calcium release by discharging mitochondria from this destructive calcium pool. To avoid artefacts, mitochondrial parameters should be carefully controlled when ruthenium red-insensitive calcium efflux is studied.

On the state of calcium ions in isolated rat liver mitochondria. I. Ion fluxes and volume changes upon Ca2+ uptake under various ionic conditions.

As to functional consequences of Ca2+ uptake in isolated rat liver mitochondria, we simultaneously measured 3H2O and [14C]sucrose spaces, monovalent cation distribution, membrane potential and delta pH across the inner membrane, and [32P]phosphate and 45Ca2+ content in parallel incubations of different ionic composition. Without added Ca2+ and phosphate, mitochondrial matrix volume, membrane potential, and delta pH depended on the concentration and permeability of monovalent cations. Despite large differences in membrane potential, maximal Ca2+ uptake was identical under all conditions. Ca2+ uptake never provoked a volume change from which an osmotic active state of mitochondrial Ca2+ could be concluded. If matrix volume shrunk this could be totally accounted for by the loss of alkali ions exchanging for calcium ions. Even phosphate taken up in conjunction with Ca2+ was osmotically silent. Volume increases here occurring if K+ was permeabilized, solely resulted from K+ uptake, though this condition may give rise to irreversible mitochondrial damage with Ca2+ and phosphate release. As mitochondrial Ca2+ is bound, an electro-chemical equilibrium across the membrane is impossible for this ion. This has to be considered in any model describing equilibria of Ca2+ with mitochondria, though present models neglect this state of mitochondrial Ca2+.

On the state of calcium ions in isolated rat liver mitochondria. II. Effects of phosphate and pH on Ca2+-induced Ca2+ release.

At high K+ concentration, the effect of phosphate on Ca2+ uptake and release was studied in isolated rat liver mitochondria. Phosphate stimulated uptake at moderately high Ca2+ concentration, and inhibited release at high pH. At low pH, phosphate accelerated Ca2+ release. Ca2+ was released after a lag phase. The time of onset and the velocity of Ca2+ release depended on Ca2+ concentration. Ca2+ release was associated with mitochondrial swelling and destruction of the permeability barrier for sucrose and for chloride. Mg2+ inhibited Ca2+ release and the accompanying events. Ruthenium red and EGTA protected mitochondria from the destructive Ca2+ release and induced an immediate, slow release of Ca2+ and phosphate. Destructive Ca2+ release depended on the time of preincubation of respiration-inhibited mitochondria in the presence of Ca2+, prior to respiration-initiated Ca2+ uptake. The presence of phosphate and mitochondrial energization antagonized the destructive effect of calcium ions. Ca2+ release by acetoacetate also depended on pH. At pH 6.8, phosphate-stimulated Ca2+ release by acetoacetate, while it inhibited the acetoacetate effect at pH 7.6. The results suggest that an essential cause for the destruction of mitochondrial integrity is an increase in the intramitochondrial concentration of free calcium ions under the influence of phosphate.