Robert W. Fleischman

Ototoxicity of cis-dichlorodiammine platinum (II) in the guinea pig☆☆☆

cis-Dichlorodiammine platinum (II) (NSC-119 875), an agent with potent antineoplastic activity which also induces renal, intestinal, and bone marrow toxicity, was tested for ototoxic effects in guinea pigs. Ototoxicity was evaluated by the disappearance of Preyers reflex in response to pure tones of 5, 7, and 10 kHz and by histopathological evaluation of the inner ear with surface preparations or midmodiolar sections. Groups of five guinea pigs treated with 8–40 ip injections of cis-dichlorodiammine platinum (II) 1 mg/kg (5 doses/wk) or with 10–15 doses of 1.5 mg/kg developed permanent deafness and histopathological lesions with pronounced loss of outer hair cells in the lower turns of the organ of Corti. Frequently, entire rows of outer hair cells disappeared and only a few isolated hair cells remained intact. Moderate numbers of outer pillar cells were missing and occasional inner hair cells and pillar cells were also damaged. Hair cell lesions included degeneration, complete cytolysis, and replacement by phalangeal scars. Single doses of 6, 9, 12, or 18 mg/kg of cis-dichlorodiammine platinum (II) produced permanent hearing loss as early as Day 3 and a scattered pattern of outer hair cell loss on Day 4 with cytological changes similar to but less severe than those observed for multiple doses. Neomycin sulfate-treated guinea pigs used as positive controls (eight consecutive daily doses of 150–250 mg/kg) showed a similar pattern of hair cell loss as produced by cis-dichlorodiammine platinum (II). Positive controls treated with a single dose of 6-aminonicotinamide from 2.5 to 20 mg/kg, developed severe damage to both inner and outer hair cells, pillar cells, and spiral ganglia. Occasional strial atrophy was also observed.

cis-Dichlorodiammineplatinum(II) (NSC-119 875): Preclinical toxicologic evaluation of intravenous injection in dogs, monkeys and mice

cis -Dichlorodiammineplatinum(II), a compound with antineoplastic and antimitotic activity, which inhibited the synthesis of DNA and to a lesser extent of RNA and proteins, was submitted to a preclinical pharmacologic evaluation in 16 dogs, 10 monkeys, and 120 mice. The LD50 following a single dose iv in Swiss mice was 13.4 mg/kg for males and 12.32 mg/kg for females. The minimum lethal dose for the dog was a single iv injection of 2.5 mg/kg or 5 daily consecutive injections of 0.75 mg/kg and for the monkey, 5 daily doses of 2.5 mg/kg. The treatment elicited severe morbidity within 5–17 days and the time of onset was dose related. Toxic signs included severe hemorrhagic enterocolitis, severe hypocellularity of the bone marrow and lymphoid tissues and marked renal lesions (renal tubular necrosis in the dog and nephrosis in the monkey). Dogs also showed occasional pancreatitis and monkeys myocarditis and occasional degeneration of the spermatogenic cells. Renal lesions were the most severe toxic changes in survivors and were manifested by azotemia, hypochloremia, proteinuria, appearance of urinary erythrocytes, leukocytes and decreased 24-hr excretion of lactate dehydrogenase in dogs and by protracted polyuria in monkeys. Occasional hypocalcemia also occurred. In dogs and monkeys that survived the treatment, toxicity regressed entirely within 55–124 days. Treatment affected primarily tissues with a high rate of cell division (intestines, bone marrow, lymphoid tissues and occasionally testes) and tissues known for primary excretion of the compound (intestines and kidneys).

Bleomycin-induced interstitial pneumonia in dogs

The intravenous administration of Bleomycin to 10 dogs at different dosages resulted in varying degrees of interstitial pneumonia in all cases and a lower incidence of nephrosis, foot pad excoriation and ulceration, onychoptosis, and alopecia. Pulmonary changes did not occur as a simple dose-related phenomenon. The lesions required at least 38 days to become apparent and appeared to increase in severity with time. Even at the lowest dose used (0·625 mg/kg body weight) very severe changes were seen 128 days after cessation of therapy. Morphological features of interstitial pneumonia were subpleural localization, focal mesothelial hyperplasia, marked hyperplasia and metaplasia of type II pneumocytes, fetalization of alveoli, and a pleomorphic inflammatory infiltrate. In cross-sections of lung lobes selected for histology approximately 1 to 22% of the parenchyma contained lesions. Involved areas showed marked elastosis, excess of reticular fibres, fibrosis, and increased acid mucopolysaccharides. The administration of Bleomycin produced pulmonary changes similar in many respects to those reported in busulphan-treated patients and desquamative interstitial pneumonia. The finding of interstitial pneumonia and pulmonary fibrosis in dogs treated with low doses over prolonged periods points the need to monitor pulmonary function in humans treated with Bleomycin.

Oral Δ9-tetrahydrocannabinol toxicity in rats treated for periods up to six months

Abstract The chronic toxicity of Δ 9 -tetrahydrocannabinol ( Δ 9 -THC) given orally 28, 90, and 180 days to Fischer rats at doses of 2, 10, and 50 mg/kg was investigated. Some 180-day treated animals were monitored after a 30-day recovery interval. The lower doses used corresponded to the Δ 9 -THC content of marihuana or hashish. In the first 10 days CNS-depression, incoordination, ataxia and passivity, poikilothermia, and hypopnea occurred to which tolerance developed. During days 10–20, irritability, hypersensitivity, hyperactivity, and aggression predominated. Fighting occurred between days 20–100. Tremors and clonic convulsions appeared after day 70 in 50% of the animals at 50 mg/kg and 12% at 10 mg/kg. Tolerance developed to CNS-stimulation, fighting and neurotoxicity and lethal cumulative toxicity was seen although the cause of death was not established. Growth rate of both sexes was inhibited despite an elevation in food consumption after a transient anorexia No morphological changes could be ascribed to Δ 9 -THC. Except for a rise of 28–45% in SGOT and 46–69% in SGPT in males at higher doses and a belated hyperglycemia in both sexes, clinical chemistry, hematological, and urinalysis parameters were within normal ranges. The greatest changes were seen in ratios of organ/FBW at 50 mg/kg: 10–20% increase in brain, lungs, kidneys, heart, and liver; 45% increase in adrenals; 96% increase in male pancreas and 13–25% increase in testis and prostate. The present investigation implicated reasonable doses of Δ 9 -THC in undesirable behavioral changes highlighted by fighting aggression, convulsive activity, and lethal cumulative toxicity. The absence of morphological changes despite changes in growth rate and organ weights indicated a functional impairment that was not an immediate threat to the life of the organism because of initiation of as yet unknown protective mechanisms.

Oral and intravenous toxicity of Δ9-tetrahydrocannabinol in rhesus monkeys☆☆☆

The toxicity of Δ9-THC was evaluated in 35 rhesus monkeys treated acutely po or iv; in 28 monkeys treated po for 28 days with 0, 50, 250 or 500 mg/kg/day; or in 16 monkeys treated iv for 28 days with 0, 5, 15 or 45 mg/kg/day. The high subacute doses selected from acute toxicity studies were chosen to establish toxicity, not to simulate human dosages. No deaths occurred in monkeys treated acutely po with up to 9000 mg/kg, but all monkeys treated acutely iv with 128 mg/kg or more died from respiratory arrest and cardiac failure. In the subacute oral study 2 of 8 monkeys treated with 500 mg/kg/day became moribund on days 10 and 14, and 1 of 6 monkeys treated with 50 mg/kg/day became moribund on day 16. Primary pathologic changes in these 3 moribund monkeys included atrophy of the pancreas, hemorrhagic ulcerative colitis, myeloid hyperplasia of the bone marrow, vacuolar nephrosis and severe serum electrolyte imbalance. All other monkeys were normal at necropsy. In the subacute iv trials, 2 of 4 monkeys treated with 45 mg/kg/day died on days 8 and 19 as a result of acute hemorrhagic pneumonia, but injection site edema, necrosis, ulceration and fibrosis also occurred. Behavioral and physiologic changes were similar in both studies and included lethargy, huddled posture, bradypnea, hypothermia, bradycardia, weight loss, anorexia and constipation. Tolerance and cumulative toxicity were also similar for the 2 studies, but monkeys treated iv showed dose-related anemia and increased BSP retention while no blood changes occurred in monkeys treated po. Pathologic changes in deceased monkeys were associated with the route of administration.

Toxicity of short-term administration of cannabinoids to rhesus monkeys.

Abstract Because of potential use as therapeutic agents, additional toxicologic studies on pure cannabinoids and cannabis extracts were conducted. Monkeys received a single iv dose of cannabidiol (CBD) or cannabichromene (CBCH) as an aqueous emulsion and LD50s were 212 and 270 mg/kg, respectively. The larger doses of CBD elicited tremors, convulsions, hypopnea, bradycardia, and cardiac failure. Survivors from smaller doses recovered in 1–3 days and liver weights increased 19–142%. CBCH evoked a dose-related hyperpnea and salivation initially but subsequent CNS inhibition resulted in apnea and bradycardia. Liver weights were elevated 8–71% and kidney weights 10–155% and pulmonary irritation, nephritis, increased BUN, and decreased serum electrolytes, hemoglobin, and RBC were found. Hashish oil containing 11.6 or 31.1% Δ9-THC caused muscle spasms, salivation, dyspnea, arrhythmia, and hypothermia and the LD50s were 326 and 435 mg/kg, respectively. Survivors recovered in a few days; lung and spleen congestion were common. Five day iv treatment with hashish oil 65–260 mg/kg) initiated similar toxicity and tolerance developed by Day 4. Growth rates were inhibited 7–20% and liver, kidney, and heart weights increased 20–100%. Delayed lethality occurred in two monkeys; histopathology included injection site necrosis, hemorrhagic foci in lungs, heart, intestines, and endocrines. Ninetyday oral treatment with CBD (30–300 mg/kg) had little effect but liver and kidney weights rose 13–56% above controls without morphologic changes. Decreased testicular size and inhibition of spermatogenesis occurred. The temporal pattern of adverse responses differentiated CBD, CBCH, and hashish oil.

Effects of cannabis on lungs.

Because of reported changes in lung function and morphology in cannabis users and animals exposed to marihuana smoke, a rat model was developed for standardized automatic chronic exposure to marihuana smoke. The smoking apparatus was regulated to present a 50-ml puff volume simultaneously from each of 3 NIDA cigarettes in a 2-sec puff for a 30-sec exposure interval followed by 30-sec of fresh air each min. Estimated Δ9-THC doses in marihuana smoke were 0.4–5 mg/kg and were relevant to those used by man. The doses were attained by varying the number of puffs from 4 to 16 which correlated with carboxyhemoglobin levels between 15 and 48% and plasma Δ9-THC levels of 34–313 ng/ml. The latter were similar to those found in human cannabis consumers (20–300 ng/ml). Behavioral signs of CNS-inhibition and CNS-stimulation were in accord with dosages and development of tolerance was observed. No pulmonary pathology could be attributed to marihuana smoke when exposure was for 14–57 days. In contrast, exposures for 87–360 days induced a dose-related focal alveolitis or pneumonitis which progressed from extensive mobilization of alveolar macrophages and foreign body cell inflammation to pronounced focal proliferative aberrations associated with focal granulomatous and cholesterol clefts. In all chronic studies, the effects of marihuana smoke were distinguished from those of placebo and tobacco smoke. The lung pathology was due to a combination of non-specific smoke particulates and cannabinoids.

Toxic effects of hexachlorobenzene after daily administration to beagle dogs for one year

Abstract Hexachlorobenzene (HCB) was administered daily in gelatin capsules to male and female beagles at 1000, 100, 10, and 1 mg/dog for 12 months. Mortality, anorexia, and weight loss occurred at the highest and, to a lesser degree, at the next lower dose. After 3 months, body weight stabilized or losses were regained. Clinical laboratory changes in severely affected animals which may have been related to malnutrition included anemia, hypoglycemia, hypocalcemia, and testicular degeneration. A dose-related neutrophilia appeared in the animals receiving the two high dosages. The most widespread pathological lesions were confined to the abdomen and included serositis, necrosis, fibrosis, and steatitis of the omentum. Nodular hyperplasia of gastric lymphoid tissue was found in all treated dogs including those at 1 mg/day (6.5–10.0 mg/kg). Four severely affected animals at the highest dose showed a generalized vasculitis and one had amyloidosis. One dog from each of the two highest doses had bile duct hyperplasia and pericholangitis. Bile and perirenal fat showed a time- and dose-related accumulation of HCB. No hepatic fluorescence was found at necropsy, indicating that these dogs were free of porphyria. Nevertheless, hepatic fluorescence was produced in female rats by feeding the same material used in the canine study.

Chronic marihuana inhalation toxicity in rats

Abstract Exposure of rats to marihuana or placebo smoke for periods of up to 87 days was performed with an automatic inhalator. Δ9-Tetrahydrocannabinol (Δ9-THC) concentrations in the marihuana smoke were similar to those inhaled by man and were presented to rats in a 50-ml puff volume of 2-sec duration and a 30-sec exposure interval followed by a 30-sec period of fresh air each minute. By varying the number of puffs from three simultaneously smoked marihuana cigarettes (2.1% Δ9-THC), 8–10 Fischer rats simultaneously received a single daily Δ9-THC dose of 0.7, 2, or 4 mg/kg, 6 consecutive days per week for 27, 57, or 87 days. Lethal cumulative toxicity in male rats began in the first week, which eventually resulted in 60% deaths, at two peak intervals, in weeks 3 and 8. During the first week, some dose-related CNS inhibition, hypothermia, and hypopnea occurred to which tolerance developed at different rates. In the second and third weeks, CNS stimulation was prevalent at lower doses. Neurotoxicity (“popcorn” reaction) occurred in 70% of both sexes on the high dose and reached a peak in weeks 3 and 8. Tolerance to these manifestations developed in subsequent weeks. Generally, growth rates and food intake decreased in males treated with the high dose but, in the other treated groups, food and water consumption were evaluated throughout the study. Increased organ to body weight ratios for the brain, lungs, and heart of males reflected decreases in final body weight. No convulsive episodes were noted. In weight-decreased rats, congestion in major organs and pulmonary edema suggested circulatory failure. In the treated animals, which were sacrificed, a dose-related moderate focal pneumonitis, characterized by the accumulation of aggregates of yellow-brown, sudanophilic alveolar macrophages, polymorphonuclear leukocytes, and lymphocytes, was observed. Female rats evoked as yet unknown protective mechanisms more efficiently than males in the face of neurotoxicity and morphological changes.

Pulmonary pathologic changes in rats exposed to marihuana smoke for 1 year.

Exposure of rats to marihuana or placebo smoke for periods up to 365 days was performed with an automatic inhalator. Δ9-Tetrahydrocannabinol (Δ9-THC) concentrations in the marihuana smoke were similar to those inhaled by man and were presented to the rats in a 50-ml puff volume of 2-sec duration and a 30-sec exposure interval followed by a 30-sec period of fresh air each minute (1 puff/min). By varying the number of puffs from three simultaneously smoked marihuana cigarettes (0.9 to 1.2% Δ9-THC), 8 to 10 Fischer rats simultaneously received a single daily Δ9-THC dose of 0.4, 0.8 or 1.5 mg/kg, 6–7 days per week for 365 days. All treatment groups contained 30 males and 30 females except for the high-dose group which had 50 males and 50 females. At each of the lower doses 260 (3%) of the animals died while at the higher dose 18108 (17%) died. Twenty-five percent (1560) of the placebo-smoked rats died primarily from carbon monoxide poisoning. No sham-smoked rats died. In deceased marihuana-smoked rats, organ congestion and focal petechial hemorrhages in the brain suggested circulatory failure. In contrast with an earlier 87-day inhalation study, pulmonary irritation progressed beyond a dose-related focal alveolitis or pneumonitis with the accumulation of yellow-brown alveolar macrophages admixed with a few neutrophils and mononuclear cells, to a spectrum of more pronounced inflammatory and focal proliferative changes. The development of focal granulomatous inflammation in the lung with giant cell forms of macrophages and cholesterol-like clefts were striking new developments in the marihuana-smoked rats, especially since these effects were dose related and nonreversible after a 30-day recovery period.