Heterotransplantation of canine sarcoma cells (BLU284) in athymic (nude) mice.

Undifferentiated canine sarcoma cells from a primary lesion were implanted subcutaneously in athymic (nude) mice. Tumors were implanted at 2 sites in each of 4 mice, and 6 of the 8 inoculation sites developed into tumors. Tumors grew and invaded surrounding tissues, as shown by histologic examination. Karyotype analysis verified that tumors were of canine origin. Cells from this tumor were serially heterotransplanted 26 times without consistent change in growth rate. Once established in nude mice, samples from 2 generations of this canine sarcoma cell line were stored cryogenically and were implanted into nude mice. Over 26 generations, 93.7% of fresh implants developed into tumors. Preliminary screening of antineoplastic drugs indicated that this tumor line was sensitive to cyclophosphamide and vincristine. The difference in sensitivity of this heterotransplanted tumor in nude mice toward the various antineoplastic agents provides a useful model for the elucidation of biochemical bases of antineoplastic drug resistance in tumors.

Inhibition of mitochondrial NADH oxidase, succinoxidase, and ATPase by naturally occurring flavonoids.

A structure-activity investigation of the inhibition of beef heart mitochondrial NADH oxidase and succinoxidase and rat liver mitochondrial ATPase by flavonoids was conducted. NADH oxidase was the most sensitive to inhibition by flavonoids: 13 of the 18 flavonoids tested inhibited NADH oxidase, whereas only 4 and 5 flavonoids inhibited succinoxidase and ATPase, respectively. The flavonoids possessing a catechol or pyrogallol moiety, and a 2,3-double bond and a 3-hydroxyl group were the most inhibitory towards the respiratory chain enzymes. The catechol or pyrogallol moiety did not exert preferential activity towards the oligomycin-sensitive ATPase because morin, which contains a meta-dihydroxy configuration, was the most potent ATPase inhibitor.

Relationship between inhibition of mitochondrial respiration by naphthoquinones, their antitumor activity, and their redox potential.

The physicochemical properties of a series of 1,4-naphthoquinones were correlated with their activities against Sarcoma-180 by Hodnett et al. [J. med. Chem. 26, 570 (1983)]. Redox potential was the most important molecular parameter determining antitumor activity in this series of compounds, suggesting that interference with electron transport contributes to their cytotoxicity. We evaluated this same series of quinones for their abilities to inhibit the beef heart mitochondrial succinoxidase and NADH-oxidase enzyme systems. They exhibited a broad range of inhibitory potencies. There was a strong relationship between succinoxidase inhibition, antitumor activity (T/C ratio), and redox potential. The redox potentials of the quinones which inhibited succinoxidase lay within the narrow range of endogenous components of the respiratory chain. In contrast, inhibition of NADH-oxidase was related to redox potential but did not significantly predict antitumor activity. These results suggest that inhibition of mitochondrial succinoxidase may be a useful preliminary screen for antitumor activity.

Metabolic consequences of dietary 2,3-dichloro-1,4-naphthoquinone (CNQ) in the rat. Alteration in anti-oxidant enzyme activities.

Dietary exposure of rats to a high concentration of 2,3-dichloro-1,4-naphthoquinone (CNQ) (2 g/kg diet) for 60 days altered cardiac mitochondrial function and activities of anti-oxidant enzymes in hepatic and cardiac tissue. CNQ moderately depressed the cardiac mitochondrial respiratory control ratio (RCR) to 85% of control; this was exacerbated to 60% of control in animals fed alpha-tocopherol-deficient diets. Dietary CNQ increased hepatic superoxide dismutase (SOD) and catalase activities and increased cardiac SOD activity, but depressed cardiac glutathione reductase and hepatic glutathione peroxidase activities. These effects are consistent with previous in vitro findings that CNQ induces oxidative stress. No significant differences in heart weight or body weight were observed in rats fed CNQ as compared to untreated controls.

Heterotransplantation of equine carcinoma cells in athymic (nude) mice.

Nondifferentiated equine carcinoma cells from a primary lesion were implanted subcutaneously in athymic (nude) mice. The cells were implanted at 2 sites each in 3 mice. At 1 of the 6 inoculation sites, a tumor developed, which invaded surrounding tissues, as shown by histopathologic examination. Karyotype analysis verified that the tumor was of equine origin. Cells from this tumor were serially heterotransplanted 20 times without change in growth rate. Once established in nude mice, this equine carcinoma cell line was stored cryogenically and then was successfully reimplanted into nude mice. All of the implants developed into tumors, over 20 generations. Preliminary screening of antineoplastic drugs indicated that this tumor line is sensitive to cyclophosphamide. Because of its ease of handling and high reimplantation efficiency, this tumor line should prove useful in equine cancer research.

Ethanol drug interaction with chlordiazepoxide and pentobarbital.

Drug interactions between ethanol and pentobarbital and ethanol and chlordiazepoxide were investigated utilizing mice. At the peak of oral ethanol (0-4 g/kg), either sodium pentobarbital (1-120 mg/kg) or chlordiazepoxide hydrochloride (2-400 mg/kg) was given intraperitoneally. Blood concentrations of ethanol, pentobarbital, chlordiazepoxide, and its pharmacologically active major metabolites were monitored utilizing either gas chromatography or high performance liquid chromatography. Lethality and loss-of-righting reflex were measured as indexes of behavioral drug interactions. It was evident from the isobolographic plot that the interactions between ethanol and pentobarbital and ethanol and chlordiazepoxide were more than additive. Interaction between ethanol and pentobarbital was greater than that between ethanol and chlordiazepoxide. Furthermore, with increasing ethanol pretreatment the shift in dose-response curves for the loss-of-righting reflex was affected more than the shift in dose-response curves for lethality. Blood concentration monitoring of each drug indicated that the rate of biotransformation of pentobarbital was significantly decreased; sequential biotransformation of chlordiazepoxide was also altered, resulting in a large accumulation of demethylchlordiazepoxide in the blood.

The isolated human pectinate muscle: a reliable preparation of human cardiac tissue.

We have developed an anatomically and functionally intact preparation of isolated pectinate muscles from readily-available surgical specimens of human right atrial appendage. Individual pectinate muscles (2-3 per specimen) are dissected free and mounted in a tissue bath. Field stimulation is used for electrical pacing, and isometric contractions are recorded. The pectinate muscle develops a stable and large force of contraction and hence is superior to strips cut from atrial appendage specimens. Isolated pectinate muscles develop spontaneous beating or can be induced to beat spontaneously by brief periods of electrical pacing or transient exposure to epinephrine or histamine. Spontaneously-beating muscles increase their rate and force of contraction in response to drugs which have positive chronotropic and inotropic effects in the whole heart. Because the force of contraction of the pectinate muscle is a function of the rate of beating, inotropic effects of agents should also be evaluated in preparations which are electrically paced at a constant rate.

Dysrhythmias caused by histamine release in guinea pig and human hearts.

Histamine is released into the systemic circulation during anaphylaxis, by drugs and by surgical procedures. Studies in animal models have conclusively demonstrated that released cardiac histamine is a major mediator of arrhythmias that occur during anaphylaxis and following the administration of histamine-releasing drugs. Several lines of evidence suggest a similar arrhythmogenic role for cardiac histamine in humans: (1) The human heart is rich in histamine; (2) cardiac histamine can be readily released from human heart in vitro by therapeutic concentrations of drugs; (3) histamine has potent arrhythmogenic effects on the human heart in vitro. Arrhythmogenic effects of histamine include enhancement of normal automaticity, induction of abnormal automaticity, induction of triggered tachyarrhythmias, depression of atrioventricular conduction, and increase in the vulnerability of the ventricles to fibrillation. A combination of H1 and H2 antihistamines is needed to block the arrhythmogenic effects of histamine. Certain arrhythmogenic effects of histamine (e.g. induction of slow responses and delayed afterdepolarizations) can also be blocked by drugs which inhibit the influx of cations through slow channels. In contrast, the commonly-used drug digitalis potentiates the arrhythmogenic effects of histamine. We propose that histamine release produced by drugs and surgical procedures may be an overlooked factor in fatal cardiac arrhythmias. Experimental studies suggest that selective pharmacological methods can be developed to block the arrhythmogenic effects of histamine.

Comparison of ethanol and barbiturate physical dependence.

The physical dependence and tolerance characteristics of barbiturates and ethanol were compared. One group of cats was given anesthetic doses of pentobarbital chronically to produce severe physical dependence ("high dose" group). Two other groups of animals were given barbital or ethanol at "chronically equivalent" doses which produced gross ataxia without anesthesia ("low dose" groups). The doses required to produce the preset level of central nervous system depression progressively increased in all three groups during the chronic administration. Functional tolerance estimated by measuring the drug concentration in the blood at the peak of the drug response was demonstrated in all three groups. Drug administration was terminated after various durations and withdrawal was rated. Severity of withdrawal was assessed by monitoring spontaneous convulsions and by rating motor, autonomic and behavioral signs. These ratings were used to compute peak intensity of withdrawal. The number of convulsions, incidence of lethality during withdrawal and peak withdrawal intensity ratings were considerably higher in the ethanol groups than in the low dose barbiturate groups. Similarly, the number of convulsions, incidence of lethality during withdrawal and peak intensity ratings were consistently higher in the high dose barbiturate groups than in the low dose barbiturate groups. The results indicate that ethanol produced more severe withdrawal than barbiturate when the level of chronic intoxication was comparable in the two groups. Also, the level of central nervous system depression during administration was an important factor influencing intensity of barbiturate withdrawal.