Anthony M. Guarino

Effect of partial hepatectomy on the responsiveness of microsomal enzymes and cytochrome P-450 to phenobarbital or 3-methylcholanthrene

Abstract The effects of phenobarbital and 3-methylcholanthrene (3-MC) pretreatment on liver weight, microsomal protein, cytochrome P-450 content, microsomal aniline hydroxylase, hexobarbital hydroxylase and P -nitroanisole O -demethylase activities have been studied in control, sham-operated and partially hepatectomized rats. In unoperated rats, phenobarbital pretreatment significantly increased liver weight, microsomal protein, P-450 content and enzyme activities toward all three substrates. 3-MC pretreatment significantly increased liver weight, P-450 content and p -nitroanisole O -demethylation without influencing the other parameters. When administered to shamoperated or partially hepatectomized rats, phenobarbital and 3-MC caused substantially the same effects, though the magnitude of the effects was less than in unoperated animals. Thus, significant increases were seen in the various parameters after injection of phenobarbital or 3-MC into hepatectomized rats. It is concluded that the regenerating liver, like the fetal or newborn liver and certain rodent hepatomas, although exhibiting low levels of microsomal enzymes, has the capacity to respond to the enzyme inducers, phenobarbital and 3-MC. Differential comparison of the enzyme changes expressed in terms of microsomal protein and also in terms of microsomal P-450 content revealed that the activity of the enzymes catalyzing p -nitroanisole and aniline oxidations parallel the microsomal P-450 content more closely than does the enzyme catalyzing hexobarbital hydroxylation.

Subcellular localization of cis-dichlorodiammineplatinum(II) in rat kidney and liver☆

The subcellualr localization of cis-dichlorodiammineplatinum(II) (cisplatin), an anticancer agent, was analyzed in rat kidney and compared to that in liver over a period of 72 hr. Male F-344 rats were given a dose of cisplatin (7 mg/kg), ip, and five subcellular fractions were isolated by differential centrifugation. Based on atomic absorption spectrometry of platinum, it was found that the kidney accumulated a greater concentration of cisplatin with a longer biological half-life than the liver. On a subcellular level, cisplatin showed a significantly high affinity for nuclei and microsomes in the kidney but not in the liver. At 24 hr, platinum concentrations were 550 ng/mg protein in the nuclear fraction (14% of total platinum) and 420 ng/mg protein in the microsomal fraction in the kidney. The cytosol contained the largest quantity of platinum (∼70%) in the kidney, of which about 40% was precipitable by trichloroacetic acid. However, the specific concentration of platinum in the acid precipitate of the cytosol was less than that of nuclei or microsomes in the kidney. Renal mitochondria and plasma membranes were associated with low concentrations of platinum. These results suggest that nuclei and microsomes may be primary target sites for cisplatin in the kidney.

Photolytic degradation of adriamycin

Adriamycin (doxorubicin hydrochloride, NSC 123 127) (ADR), an antibiotic widely used in cancer chemotherapy (Tan et al 1973; Blum 1975), consists of an anthracycline moiety and an amino sugar, daunosamine, linked together through a glycosidic bond. Like related anthracycline antibiotics, ADR is believed to be photosensitive. However, no photolytic studies on this group of compounds have been published, and warning of their possible photodegradation has been reported only in the clinical brochure offered by the manufacturer. This report concerns the kinetics of degradation of ADR solutions when exposed to fluorescent light. ADR bulk powder was obtained from the Division of Cancer Treatment, NCI, NIH, and was chromatographically purified (>95 %) before use (Tavoloni & Guarino 1980). ADR solutions in various solvents were prepared in Pyrex-glass scintillation vials closed tightly with plastic caps. For each solvent, 6 ADR concentrations ranging from 10 to 500 pg ml-1 were prepared. All samples were prepared in triplicate and protected from light with aluminium foil before each study. The ADR concentration of each solution was determined fluorometrically (Bachur et al 1970), and A D R photodegradation, resulting from breakdown of the naphthacenequinone nucleus, by measuring the loss of fluorescence over time. In all instances, fluorescence values were determined in triplicate and the mean for each determination recorded. The photolysis of ADR was studied under three different experimental conditions. For each set of triplicate vials, one was kept in absolute darkness, one exposed to room light and one irradiated with intensive light. Vials of ADR solutions kept in the dark remained covered with aluminium foil and stored in a dark room. In room light studies, vials were kept upright on a table in the laboratory and light was provided by 12 40-watt ceiling fluorescent-tubes, mounted approximately 2 rn above the vials. In intensive light studies, samples were exposed to room light as above, and simultaneously irradiated with 2 additional desk lamps, each equipped with two 15-watt tubes, positioned on either side of the vials 10-20 cm from the samples. Samples were assayed for fluorescent activity at different intervals for 144 h. Where photodegradation of A D R was very rapid, fluorescence was determined as often as every 2 min, whereas in other more stable solutions fluorometric determinations were made a t 3, 6, 12 or 24 h intervals.

Mouse and large-animal toxicology studies of twelve antitumor agents: Relevance to starting dose for Phase I clinical trials

SummaryLarge-animal toxicology is presently used to establish a starting dose for clinical trials with new cancer chemotherapeutic agents. The relevance of dog, monkey, and mouse data for Phase I clinical trials has been retrospectively analyzed with twelve diverse agents (chlorozotocin, maytansine, anguidine, tritylcysteine, piperazinedione, Bakers antifol, thalicarpine, 3-deazauridine, gallium nitrate, cis-dichlorodiammineplatinum(II) (DDP), 4′-(9-acridinylamino)methanesulfon-manisidide (AMSA), and N-phosphonacetyl-l-aspartic acid (PALA). Schedules studied clinically included a daily x 5 schedule and a single dose schedule (three drugs), a daily x 5 schedule only (three drugs), and a single dose schedule only (six drugs). One-thrid of the toxic dose low (TDL) in the most sensitive large-animal species (dog or monkey), expressed in mg/m2, was a tolerable starting dose in humans in all instances for the schedules employed. The number of dose escalation steps to reach the human maximum tolerated dose (MTD), according to the commonly used Fibonacci dose escalation scheme, varied from 2 to more than 12. Had one-third the LD10 in mice, expressed in mg/m2 been applied, this would also have yielded safe starting dose levels, and would actually have required a lesser number of dose escalations to reach the human MTD. This analysis confirms that mouse data may be quite useful in determining safe starting doses for Phase I trials with anticancer chemotherapeutic agents.

Pharmacokinetic modeling of the dogfish shark (Squalus acanthias): Distribution and urinary and biliary excretion of phenol red and its glucuronide

A flow-limited multicompartment model simulates the distribution and disposition of phenol red in the dogfish after intravenous administration. Glucuronide conjugate, as well as parent compound, was found in urine and bile, but not in plasma, kidney, or liver tissue. An apparent 4-hr lag in phenol red appearance in the gall bladder was simulated using two stirred tanks connected in series to represent the bile ducts. The model should facilitate use of the dogfish in pharmacokinetic studies of drugs, environmental contaminants, and other xenobiotics.

Disposition and Metabolism of Adriamycin in the Rat

Adriamycin (ADR) is extensively excreted in bile and moderately in urine following its administration to anesthetized rats. At intravenous doses ranging from 5 to 20 mg/kg, approximately 34% of the injected ADR is excreted in bile as total drug equivalents and 6-8% in urine over a 10-hour period. Thin-layer chromatography of bile, urine and tissue extracts revealed the presence of three major metabolic products in addition to the parent drug. In either bile or urine, unchanged ADR was the predominant form excreted and accounted for about 70% of the total drug equivalents. Adriamycinol and ADR conjugates were the major metabolites, were excreted at comparable rates and together accounted for most of the remaining fluorescence or radioactivity in either body fluid. ADR itself was the main form found in all tissues examined. ADR conjugates were not detected in any tissue whereas adriamycinol was observed in kidney, heart and spleen, but not in the liver. ADR aglycones could not be detected in the heart. They appeared preferentially in the liver where, 3 h after ADR was injected, they accounted for about 40% of total tissue fluorescence or radioactivity. The low rate of ADR conversion observed in the present studies supports the hypothesis of species difference in the metabolism of the drug.

Biliary and urinary excretion of adriamycin in anesthetized rats.

Adriamycin (ADR), an antibiotic widely used in cancer chemotherapy, is rapidly cleared from plasma, extensively excreted in bile and only moderately in urine during the first few hours after its intravenous injection to anesthetized rats. When bile and urine are collected in bile duct- and bladder-cannulated rats, about 33-35% of the injected doses, 5, 20 or 40 mg/kg ADR, is excreted in bile as total drug equivalents during a 10-hour collection period, while 4-8% is eliminated in urine. Within 60 min from the injection, as much as 15-17% of the dose is excreted in bile and 1-3% in urine. The biliary excretion of ADR, within the dose range of 5-40 mg/kg, is not saturable, is linearly related to the dose administered, and occurs in absence of choleretic or cholestatic manifestations. Conversely, the urinary excretion of the drug is a dose-limited process; when ADR is injected at 40 mg/kg, a significantly lower percentage is eliminated in urine. This decline is associated with a severe, although transient, antidiuretic effect thus suggesting an intrinsic toxicity of the drug at this high dose on renal function.

Enzymic and biochemical composition of smooth and rough microsomal membranes from monkey, guinea pig and mouse liver.

Abstract Smooth and rough microsomal membranes, prepared by density gradient centrifugation from livers of mice, guinea pigs and monkeys, were compared on the basis of biochemical composition, enzyme distribution, and fine structure. Phospholipid and cholesterol distribution varied considerably between smooth and rough membranes and between animal species. The following components of microsomal drug metabolism were studied: ethylmorphine demethylase, l -benzphetamine demethylase, aniline hydroxylase, NADPH cytochrome c reductase, cytochrome P-450, and spectral changes resulting from microsome-substrate interaction. Expressed per milligram of protein, all these components were concentrated about 2-fold in the smooth membranes of monkey and guinea pig liver, but were approximately evenly distributed between smooth and rough membranes of mouse liver. The results support the concept of heterogeneity among microsomal membranes. In addition to the well recognized species differences in microsomal drug metabolism, our data indicate important species differences in submicrosomal membrane composition and enzyme distribution.

Tissue distribution of [14C]DDT in the lobster after administration via intravascular or oral routes or after exposure from ambient sea water

Abstract A pharmacokinetic approach to studying the fate and distribution of [ 14 C]DDT was employed using the lobster, a species of obvious economic importance. The radioactive pesticide was administered by 3 different routes; intravascular, oral and by exposure from the ambient water. After intravascular administration there was very rapid removal of [ 14 C]DDT from the plasma accompanied by a strikingly persistent increase in the amount of radioactivity in the hepatopancreas. Most (>90%) of the radioactivity in this organ was shown by TLC to be the parent pesticide. Seven days after injection of [ 14 C]DDT approximately 90% of the administered radioactivity was found in the hepatopancreas and the concentrations in this organ decreased with a t 1 2 of 46 days. One month after treatment with 0.1 mg/kg of [ 14 C]DDT, the only other organs which contained more than 1% of the administered dose were egg masses and muscle. When the pesticide was administered to the lobster from ambient water or from food, the hepatopancreatic compartment again dominated, with more than 90% of the absorbed dose occurring in this organ 7 days after treatment. Studies conducted of residue levels in untreated lobsters indicated that the egg masses contained the largest concentration of total DDT metabolites (1 ppm). The hepatopancreas contained about 0.4 ppm while the carcass (muscle) contained about 0.1 ppm. These distribution studies suggest that while the lobster may protect itself from DDT toxicity by sequestering the pesticide in the hepatopancreas and in egg masses, bioconcentration in these tissue could be hazardous to species consuming these parts of the lobster.

In vitro drug metabolism in male and female athymic, nude mice.

Abstract Drug metabolism was studied in hepatic microsomal and post microsomal supernatant fractions from male and female athymic nude mice (nu/nu) and heterozygous (+/nu) and homozygous (+/+) wild-type controls. In males, the following enzyme activities were higher in athymic mice than in the wild-type: NADPH cytochrome c reductase, ethylmorphine and aminopyrine N-demethylases, native UDP glucuronyltransferase, and glutathione (GSH) S-aryltransferase. No differences were observed between groups in UDPNAG-activated UDP-glucuronyltransferase, N-acetyltransferase, or aniline hydroxylase activities or in amounts of cytochrome P-450. In female athymic mice, only ethylmorphine and aminopyrine N-demethylase activities were significantly higher than in female wild-type controls (+/+). The female athymic mice had mixed function oxidase activities that were less than the male athymic mice. There were no sex or strain differences in response to treatment with phenobarbital or 3-methylcholanthrene.