An experimental rat model of local bone cancer invasion and its responsiveness to ethane-1-hydroxy-1,1-bis(phosphonate).

Line A Walker carcinoma differs from line B in that it does not elicit hypercalcemia and hypercalciuria when implanted in rats at various sites (s.c, i.m., intraaortically). However, Walker 256/A, unlike line B, may invade the tibia when implanted i.m. in the adjacent gastrocnemius muscle. This invasion was evaluated by measuring the increased weight of the bone and decreased calcium concentration per unit weight of the tibia, by reduced opacity to X-ray, and by the presence of tumor cells in the compact bone cortex. Ethane-1-hydroxy-1,1-bis(phosphonate), a diphosphonate derivative, at a dose of 10 to 30 mg/kg/day s.c., prevented cancer cell invasion of the tibia as judged by the above criteria. This inhibition was obtained with no apparent effect on the growth of Walker 256/A carcinoma.

Secretion of lipoproteins, apolipoprotein A-I and apolipoprotein E by isolated and perfused liver of rat with experimental nephrotic syndrome.

Nephrotic syndrome induced in the rat by the administration of puromycin aminonucleoside is accompanied by a hyperlipoproteinemia characterized by an elevation of all plasma lipoproteins, particularly of VLDL (1.006 g/ml) and HDL1 (1.050-1.090 g/ml). The increase of HDL1 is due to the accumulation of a lipoprotein species floating mainly in the density interval 1.050-1.090 g/ml, in which apolipoprotein A-I replaces apolipoprotein E as the major constituent peptide. This lipoprotein has been designated nephrotic HDL. The present study was conducted to establish whether nephrotic liver secreted more lipoproteins than the control liver and, in addition, produced a lipoprotein similar to nephrotic HDL found in plasma. Isolated livers from control and nephrotic rats were perfused with a lipoprotein-free medium for 3 h in a recirculating system. Lipoproteins were isolated by ultracentrifugation; apolipoprotein A-I and apolipoprotein E were measured in the whole perfusate at various time intervals. Nephrotic liver secreted twice as much VLDL and HDL2 and 30% more LDL and HDL1 than the control liver. This was accompanied by an increased secretion of both apolipoprotein A-I and apolipoprotein E, the levels of which were 6.5- and 2-fold, respectively, of those found in the control perfusates at the end of the perfusion. In view of the increased secretion of apolipoprotein A-I, the apolipoprotein A-I to apolipoprotein E ratio was much higher in the perfusates of nephrotic livers than in those of the controls. The concentration of apolipoproteins A-I and E in plasma of nephrotic rats was 7- and 2-fold, respectively, of that found in the plasma of the controls. In the perfusates of the nephrotic livers, we could not find a HDL1 (1.050-1.090 g/ml) rich in apolipoprotein A-I similar to that isolated from plasma (nephrotic HDL). We suggest that the latter is formed in the circulation from the intravascular modification of HDL2 secreted in excess by the liver.

Effect of etidronate disodium on the interactions between malignancy and bone.

This study was designed to assess the capacity of etidronate disodium (etidronate) to affect neoplastic involvement of bone by murine tumors. Using sublines of the Walker 256 carcinoma, differing in the pattern of bone involvement, etidronate was found to inhibit hypercalcemia caused by systemically acting humoral factors, to inhibit bone metastasis following inoculation of tumor cells into the abdominal aorta, and to reduce the invasion of bone adjacent to tumors. Etidronate was also found to prevent bone metastasis in syngeneic rat mammary carcinoma. Etidronate was found devoid of direct antineoplastic activity, whether administered intramuscularly, subcutaneously, or intravenously, in a series of murine tumors of different histologic varieties. At the same time, it was shown that etidronate did not modify the antineoplastic activity of any of the major chemotherapeutic agents used in these studies, nor did it demonstrate any degree of immunosuppression. The experimental models used for this study should prove useful in evaluating agents that may affect the various types of bone involvement seen in neoplastic disease. The drug's apparent lack of interference with the antineoplastic activity of standard cytotoxic agents and its lack of immunosuppressive activity suggest that it may be readily adaptable to combination chemotherapy regimens.

Doxorubicin toxicity and pharmacokinetics in old and young rats.

Doxorubicin (Dx) toxicity was compared in old (24 months) and young (6 weeks) Crl:CD(SD) BR male rats, and a clear age-related increase was found. The mortality of all animals receiving a single i.v. Dx dose was followed for 270 days. Old rats died after doses of 2.5 mg/kg, while young animals died after doses two times higher, 5 mg/kg. In old rats body weight loss started 10 to 15 days after Dx, compared to 50 to 80 days for young animals. In young and old rats pharmacokinetic and metabolic studies of Dx were conducted in vivo and in the liver perfusion model. Peak levels of Dx and areas under the time/concentration curves (AUC) in serum and in several tissues of old rats were 1.5 to 2 times higher than in young rats. Concentrations of Dx metabolites in serum and tissues (doxorubicinol, Dxol, and doxorubicinone, Dxone) in young and old rats were not noteworthy. However, higher percentages of Dxone than Dxol were found in both groups in vivo and in vitro. Old livers appeared to produce more Dxone as a percentage, particularly in the bile, which was higher. Urinary elimination of Dx markedly slowed with age; only small amounts of the metabolites were eliminated in urine. In vivo and in vitro availability of Dx and its metabolites is discussed in view of their possible role in the greater toxicity observed in 24-month-old rats.

Pharmacokinetics of fotemustine and BCNU in plasma, liver and tumor tissue of rats bearing two lines of Walker 256 carcinoma.

The plasma and tissue pharmacokinetics of fotemustine (diethyl-1-[3-(2-chlorethyl)-3-nitrosoureido]-ethylphosphonate+ ++) and BCNU (1,3-bis-[2-chlorethyl]-1-nitrosourea) were investigated in healthy control rats and in animals bearing either the nitrosourea-sensitive line A (W256/A) or the nitrosourea-resistant line B (W256/B) of Walker 256 carcinoma. The antitumor activities of these nitrosoureas were similar following i.v. doses ranging from 10 to 40 mg/kg. For both drugs, the survival of tumor-bearing rats was lower in the W256/B than in the sensitive W256/A line. Some sex differences were observed, female rats being more responsive than males to both drugs. Nitrosourea concentrations were assayed in plasma and tissues by differential pulse polarography so as to assess whether the pharmacokinetics could explain the differences in antitumor activity. The antineoplastic effects of fotemustine seemed to be influenced by its pharmacokinetics. The plasma AUC of the intact nitrosourea was higher in females than in males. Fotemustine was cleared 2-5 times more slowly than BCNU from tumor tissue, and its clearance was higher in W256/B- than in W256/A-bearing rats. This suggests that the antitumor activity in the responsive line might partly be due to longer exposure of the growing tumor to the drug. The distribution volume of both nitrosoureas in plasma was higher in tumor-bearing animals than in healthy controls, indicating that the tumor tissue probably constitutes an additional distribution space.

Protective effect of diosmetin on in vitro cell membrane damage and oxidative stress in cultured rat hepatocytes.

Primary cultures of rat hepatocytes were used to study the effects of the flavonoids diosmin and its main metabolite diosmetin on the cell membrane damage caused by erythromycin estolate (EE) and oxidative stress caused by tert-butylhydroperoxide (TBHP). The damage was evaluated by the leakage of intracellular enzymes lactate dehydrogenase, aspartate-aminotransferase and the residual cell content of a lysosomal marker acid phosphatase (AP). After treating the cells for 40 h with diosmetin EE induced less enzyme leakage. The content of AP was kept higher by diosmetin pretreatment after 6 h exposure to EE. Diosmin at the same concentrations had barely any effect. Diosmetin, but not diosmin, also protected against TBHP toxicity and this was related to lower lipid peroxidation and higher glutathione content caused by pretreatment with the flavonoid. When the cells were treated simultaneously with TBHP and diosmetin after 21 h of culture, the protection by the flavonoid was even higher. In fact the antioxidant activity of diosmetin was considerably greater than that of diosmin. After 40 h exposure to both flavonoids diosmin but not diosmetin was detectable in the cell membrane fraction, suggesting that the latter's protective effect is associated with its metabolites.

Role of the hippocampus in the sex-dependent regulation of eating behavior: studies with kainic acid.

Marked hyperphagia with an increase in the rate of body weight gain was noted in adult female rats 4 days after injections of 2 nmoles of kainic acid into the dorsal and ventral parts of hippocampus. The effect was still present 70 days later. At this time the increase in daily food intake and body weight gain amounted, respectively, to 39% and 93% over the control value. There was no change in water intake. The injection of kainic acid into only one part of the hippocampus--either dorsal or ventral--did not induce hyperphagia. Male rats with kainic acid lesion did not show changes in food intake or body weight gain as compared to vehicle-treated controls. In both sexes the degeneration of hippocampal perikarya induced by kainic acid was associated with a 50-60% decrease in glutamic acid decarboxylase activity and [3H]glutamate uptake, as well as with a small decrease in [3H]glutamate uptake in the hypothalamus, an area that receives glutamatergic fibers from the hippocampus. The results show that the hippocampus appears to play an important role in appetite motivation control by a mechanism which is sex-related.

Hepatobiliary metabolism and urinary excretion of 4-demethoxydaunorubicin as compared to daunorubicin in rats.

The hepatic metabolism and biliary excretion of 4-demethoxydaunorubicin (4DDM) was studied in Crl: CD(SD) BR rats by the liver perfusion technique. In the same strains of rats urinary excretion was investigated in vivo. Daunorubicin (DM) was always included for comparison. The drugs and their metabolites were determined in the perfusion medium, in the bile and liver and in the urine by high-performance liquid chromatography with fluorimetric detection. Compared to its analogue DM, 4DDM markedly differed in the metabolic and excretory profile. The cumulative biliary and urinary excretion of 4DDM and the metabolites was quantitatively lower than that of DM (18% vs 36% of the dose) and was consistent with prolonged persistence of 4DDM in plasma in vivo. The extensive carbonyl reduction of 4DDM and DM observed in previous in vivo pharmacokinetic studies was also evident in this study. 13-hydroxy metabolites, daunorubicinol (DMol) and 4-demethoxydaunorubicinol (4DDMol), either as such or after glycosidic cleavage, i.e. 4DDMol aglycone, were present in appreciable amounts in the perfusion medium, bile, liver and urine. In the hepatobiliary system, however, the 13-hydroxy derivative of DM amounted to a much lower fraction than the DM aglycone (17% vs 50% of the total dose), 80% of the total 4DDM dose was accounted for by 4DDMol aglycone. In urine uncleaved DMol or 4DDMol represented more than 75% of the total amount excreted for both drugs. Conjugation, a major step in the excretion of aglycones, seems to play a minor role in the biliary and urinary excretion of 4DDM and 4DDMol.

Antitumor activity of the novel nitrosourea S10036 in rodent tumors.

The activity of the novel anticancer agent diethyl 1-3-(chloroethyl)-3-nitrosoureido ethyl phosphonate (S10036) was investigated on several rodent tumors. S10036 showed a good efficacy, comparable to that of the anticancer agent BCNU, against i.p transplanted P388 and L1210 leukemias. S10036 was very effective against the primary tumor and metastases of i.m transplanted M5076 reticular cell sarcoma of the mouse and against subline A of the Walker carcinoma of the rat. It was inactive against rodent tumors resistant to BCNU such as L1210/BCNU, ICIG-Ci4 murine fibrosarcoma and the Walker carcinoma subline B in the rat.

No evidence of intracellular oxidative stress during ischemia-reperfusion damage in rat liver in vivo.

The role of the intracellular generation of reactive oxygen species in the pathogenesis of ischemia-reperfusion damage of the liver was investigated in two in vivo rat models. Global hepatic ischemia was produced in the left and median lobes for 90 min followed by 60 min reperfusion to the total organ (model A) or only to the ischemic lobes (model B). Although both regimens caused significant rises in serum transaminases, this rise was higher in model B. In neither model was intracellular hydrogen peroxide production nor increased glutathione disulfide in bile found. The activities of various antioxidant enzymes were not affected by ischemia or ischemia-reperfusion. In conclusion, oxygen-free radicals are unlikely to be produced in the cells of rat liver during ischemia-reperfusion.

Comparative kinetics of benz(a)anthracene, chrysene and triphenylene in rats after oral administration. I. Study with single compounds.

Distribution and elimination of benz(a)anthracene (B(a)A), chrysene (Ch), and triphenylene (Tr) were compared after oral administration of the single compounds to 7-8-week-old female rats. These four-ring isomers were chosen because of their different carcinogenicity. The compounds have high affinity for lipid-rich tissues such as brain, mammary and parametrial adipose tissue. The relative availability of these compounds in the tissues examined decreased in the order Tr greater than B(a)A greater than Ch, but fecal elimination diminished in the opposite order Ch greater than B(a)A greater than Tr. Availability and fecal elimination of single polycyclic aromatic hydrocarbons (PAH) were influenced by the dose and concentration of PAH in the vehicle.

Effect of fasting, induction, sex and age on clearance of benz(a)anthracene and chrysene by isolated perfused rat liver.

Hepatic clearances of benz(a)anthracene (B(a)A) and chrysene (Ch) by isolated perfused liver of female rats were similar when measured with hydrocarbons singly (B(a)A 4.3 ml/min, Ch 5.1 ml/min) or in a mixture (4.7 resp. 5.3 ml/min). Clearance of both isomers by livers from 24 h fasted donors was approximately halved. In vivo pretreatment with B(a)A (22.8 mg per kg for 2 days) significantly increased elimination of both hydrocarbons. Hepatic clearance of B(a)A was significantly higher in male rats than in females of the same age (8 weeks). Elimination of both hydrocarbons was significantly lower in 2 year-old males.

Comparative kinetics of oral benz(a)anthracene, chrysene and triphenylene in rats: study with hydrocarbon mixtures.

Distribution and elimination of benz(a)anthracene (B(a)A) was compared in blood, liver, brain, parametrial adipose and mammary tissue of young female rats after oral administration of the polycyclic aromatic hydrocarbons (PAH) singly or in equimolar mixtures with chrysene (Ch) or triphenylene (Tr). The relative availability of B(a)A in tissues was not influenced by Ch or Tr in the mixture. However, the presence of B(a)A halved the relative availability of Tr and raised that of Ch. The relative availability of the three isomers decreased in the order Tr greater than B(a)A greater than Ch; this may be correlated to their solubility in water.

Metabolism of caffeine to 6-amino-5-[N-methylformylamino]-1,3-dimethyluracil in the isolated, perfused liver from control or phenobarbital-, beta-naphthoflavone- and 3-methylcholanthrene-pretreated rats.

Caffeine metabolism to 6-amino-5-[N-methylformylamino]-1,3-dimethyluracil was studied in the isolated, perfused rat liver. The [2-14C]-labelled drug and metabolites were separated by thin-layer chromatography or high-pressure liquid chromatography. The chemical structure of 6-amino-5-[N-methylformylamino]-1,3-dimethyluracil was confirmed by mass spectrometry and it was quantitatively determined by liquid scintillation counting. 6-Amino-5-[N-methylformylamino]-1,3-dimethyluracil is one of the major metabolites of caffeine found in the perfusion medium. The kinetics of caffeine elimination and of the uracil metabolite formation were studied up to 2 h perfusion time using livers from control rats and rats pretreated with phenobarbital, beta-naphthoflavone or 3-methylcholanthrene. Phenobarbital pretreatment did not modify the rate of caffeine elimination or the extent of 6-amino-5-[N-methylformylamino]-1,3-dimethyluracil formation. In contrast, there was a highly significant inducing effect on both drug elimination and formation of the uracil metabolite in perfusions of livers from beta-naphthoflavone- and 3-methylcholanthrene-pretreated animals.

Effects of chronic treatment with di-(2-ethylhexyl) phthalate on rat liver microsomal activities.

The effects of chronic di-(2-ethylhexyl)phthalate (DEHP) on liver microsomal activity were studied in rats. Daily doses of 50 and 500 mg/kg for 4 weeks did not affect O-demethylation, aromatic hydroxylation, N-demethylation, C3-hydroxylation, styrene monooxygenase, glutamic-oxalacetic and glutamic-pyruvic transaminases (GOT, GPT). Inhibition of glutathione-S-transferase A and C and induction of epoxide hydrase, glutathione-S-transferase B and nitroreductase activity were instead observed. Protein, cytochrome P-450 and reduced glutathione levels in liver did not appear to be affected by DEHP pretreatment.

Insulin-mimetic effects of vanadate in preventing the increase of P450IIIA and P450IA subfamily proteins in streptozotocin-diabetic rats.

The insulin-mimetic effects of vanadate in preventing the increase in the level and activity of several P450 proteins in streptozotocin-diabetic rats were examined, in order to extend knowledge of the insulin-like actions of vanadate from glucose metabolism to P450-dependent metabolism. The diabetic state caused by the pancreatic beta cell toxin streptozotocin results, like the diabetes of genetic origin, in major alterations in the expression of P450 isozymes. We focused our attention on the P450III and P450I isoforms, known to be altered during the onset of diabetes. We found an increase in P450IIIA1-linked erythromycin demethylase activity (to about double the control level) and in the relative levels of P450III and P450I isozymes after 2 weeks of uncontrolled diabetes. These parameters were not different from control values in rats given vanadate in drinking water for 2 weeks after streptozotocin administration or in insulin-treated rats. In summary, vanadate appears to exert insulin-mimetic actions on the P450III and P450I family proteins that have a key role in cytochrome P450-dependent metabolism.

Effects of in vivo treatment with a new fluorinated macrolide (P-0501A) and other erythromycins on drug clearance and hepatic functions in perfused rat liver.

The hepatic clearance and the effects of a new fluorinated macrolide (P-0501A) on the functions of the isolated, perfused rat liver were compared with two known erythromycins--the base and the estolate--after 7 days of treatment (1.36 mmol/kg po daily). The in vitro metabolism of the antibiotics was induced to different extent but only the base and P-0501A were cleared from the perfusate and the liver faster than in untreated animals. In untreated rats the therapeutically active form of P-0501A was excreted in the bile more than the base and the estolate; after pretreatment, biliary excretion of all erythromycins was nearly double. The content of inactive, complexed cytochrome P-450 was increased only by the base and estolate, with various effects on microsomal activities (some induced, e.g. aminopyrine demethylation, other reduced, e.g. pentobarbital clearance). The clearance and biliary excretion of sulphobromophthalein was not affected by treatment with P-0501A or the base, but was significantly reduced by estolate.

Effects of a new fluorinated macrolide (P-0501A) and other erythromycins on drug metabolizing enzymes in rat liver.

The effects of a new fluorinated macrolide (P-0501A) on drug metabolizing enzymes of rat liver were compared with three erythromycins--the base, the stearate and the estolate--after 7 days of dosing (1.36 mmol/kg po daily). The three erythromycins induced the synthesis of microsomal enzymes, but the products of their metabolism inactivated cytochrome P-450 in the order base less than or equal to stearate less than estolate. N-Demethylation of erythromycin and aminopyrine increased, while O-demethylation of 4-nitroanisole was reduced and hydroxylation of aniline was not changed after in vivo treatment. Pentobarbital sleeping time was prolonged and liver glutathione levels were lower in treated rats than in controls. In contrast to the three erythromycins, P-0501A did not induce the synthesis of microsomal enzymes, did not form an inactive complex with cytochrome P-450 and did not affect mono-oxygenase activities or pentobarbital narcosis.

Two lines of Walker carcinoma 256: their peculiarities and different interactions with the host.

Two sublines of Walker 256 carcinoma have been characterized for their ability to metastasize and to induce cachexia. The invasive, metastasizing line A induced terminal anorexia in rats with a mean survival time of 27 +/- 1.5 days. The non-invasive line B induced early anorexia and cachexia with a mean survival time of only 15 +/- 1 days. At death, the line B tumor was still smaller than the line A one, and no metastases were detectable. These two sublines are discussed as a composite model for studying anorexia and cachexia together with invasion and metastasis.

Polarographic assay of submicrogram quantities of cis-dichlorodiamineplatinum (II) in biological samples.

Differential pulse polarographic assay of the antineoplastic agent cis-dichlorodiamineplatinum II and its analogues was performed after acid oxidative hydrolysis (HClO4, HNO3, HCl) of biological samples (plasma, tissue homogenates, urine) and reaction with ethylenediamine. Platinum levels and kinetics were determined in blood and urine of patients with non-oat-cell lung carcinoma. Detection limit of the polarographic assay was 0.5 ng platinum; analytical error was +/- 3%. Levels of free cis-dichlorodiamineplatinum (II) in plasma fell in samples stored at -20 degrees C; the half-life of free drug was 38 h.