Carcinogen In Laboratory Animals Research Articles

Evaluation of the Evidence That Tobacco-Specific Nitrosamines (TSNA) Cause Cancer in Humans

Considerable evidence exists that TSNA may cause cancer in humans. Strong evidence suggests that the NNN and NNK in snuff are at least partially responsible for the excess of oral cancer among snuff dippers. Tobacco smoke, because it is a combustion product, is a far more complex matrix than is smokeless tobacco. Nonetheless, NNK is well established as a lung carcinogen in laboratory animals at levels similar to those experienced by heavy smokers. Although the epidemiology of the smoking/lung cancer association has been studied extensively and is well established as a causal relationship, few attempts have been made to determine the effective constituents in tobacco smoke, much less to quantify them and to reduce their levels in consumer products. More work is needed to determine TSNA uptake in smokers and users of smokeless tobacco and to determine the factors involved in the possible endogenous formation of TSNA and in their metabolic activation or inhibition of their formation. Exploratory work is in progress with various biological markers that may prove useful in quantifying the levels of TSNA exposure in humans. Epidemiologic evidence indicates that in addition to tobacco use other factors are causally related to the development of cancers of the oral cavity, lung and esophagus and that these factors (e.g., radon exposure in uranium miners who smoke) can act synergistically. Experimental investigation of some of these synergistic effects may prove to be a step in the direction of determining the proportion of various cancers that are likely to be attributable to various causes and ultimately to the various constituents of tobacco exposure.

In vitro studies of aldehyde effects related to human respiratory carcinogenesis

Aldehydes are ubiquitous compounds which are generated from many both endogenous and exogenous sources. Primarily because certain aldehydes are respiratory toxicants and carcinogens in laboratory animals, and also because they are present in both tobacco smoke and automotive emissions, cultured human bronchial cells have been used to study the ability of aldehydes, i.e., acrolein and formaldehyde, to cause pathobiological effects associated with carcinogenesis. Comparative studies indicate that each aldehyde distinctly affects several molecular and cellular variables including colony-forming efficiency, clonal growth rate, membrane integrity, formation of cross-linked envelopes, levels of cytosolic free calcium, low-molecular-weight thiol status, DNA structure, i.e., formation of DNA single-strand breaks and DNA-protein cross-links, and various DNA repair mechanisms. In relation to the toxicity exerted by these agents, acrolein induces differentiation more readily than formaldehyde whereas formaldehyde causes much higher levels of genetic damage than acrolein. However, for all biological endpoints measured, acrolein on a molar basis is always more potent than formaldehyde. Taken together, a variety of effects that relate to cell death, accelerated epithelial terminal differentiation and genotoxicity are associated with aldehyde exposure, which in human airways may have a role in the pathogenesis of various diseases. In the development of cancer, the possible contribution of aldehydes from both intra- and extra-cellular sources may partly depend on the ability of target cells to detoxify and counteract those aldehyde-related effects believed to critically relate to multi-stage carcinogenesis.

Use of statistical decision rules for evaluating laboratory animal carcinogenicity studies

In the evaluation of long-term rodent carcinogenicity studies, many different tumor sites and types are evaluated, which may increase the likelihood of a statistical false positive. To deal with this issue, a number of statistical decision rules have been proposed that take into account multiple comparisons. This paper discusses the various types of decision rules and evaluates the factors that may lead to different interpretations of experimental results. These concepts are illustrated by examining the statistical decision procedures used by three analysts to evaluate the results of 25 long-term rodent carcinogenicity studies carried out by the National Cancer Institute. Agreement among these decision rules is shown to be greater than originally reported. It is also concluded that while the application of statistical decision rules may be of value in some instances to guard against statistical false positives, the final interpretation of the data should be based on biological as well as statistical considerations. Thus, statistical decision rules should not be employed as a substitute for sound scientific judgment in the overall evaluation of these experiments.

Use of Statistical Decision Rules for Evaluating Laboratory Animal Carcinogenicity Studies

Use of Statistical Decision Rules for Evaluating Laboratory Animal Carcinogenicity Studies. HASEMAN, J. K. (1990). Fundam. Appl. Toxicol. 14, 637–648. In the evaluation of long-term rodent carcinogenicity studies, many different tumor sites and types are evaluated, which may increase the likelihood of a statistical false positive. To deal with this issue, a number of statistical decision rules have been proposed that take into account multiple comparisons. This paper discusses the various types of decision rules and evaluates the factors that may lead to different interpretations of experimental results. These concepts are illustrated by examining the statistical decision procedures used by three analysts to evaluate the results of 25 long-term rodent carcinogenicity studies carried out by the National Cancer Institute. Agreement among these decision rules is shown to be greater than originally reported. It is also concluded that while the application of statistical decision rules may be of value in some instances to guard against statistical false positives, the final interpretation of the data should be based on biological as well as statistical considerations. Thus, statistical decision rules should not be employed as a substitute for sound scientific judgment in the overall evaluation of these experiments.

Multiple-site carcinogenicity of benzene in Fischer 344 rats and B6C3F1 mice.

Toxicology and carcinogenesis studies of benzene (CAS No. 71-43-2; greater than 99.7% pure) were conducted in groups of 60 F344/N rats and 60 B6C3F1 mice of each sex for each of three exposure doses and vehicle controls. These composite studies on benzene were designed and conducted because of large production volume and widespread human exposure, because of the epidemiologic association with leukemia, and because previous experiments were considered inadequate or inconclusive for determining carcinogenicity in laboratory animals. Using the results from 17-week studies, doses for the 2-year studies were selected based on clinical observations (tremors in higher dosed mice), on clinical pathologic findings (lymphoid depletion in rats and leukopenia in mice), and on body weight effects. Doses of 0, 50, 100, or 200 mg/kg body weight benzene in corn oil were administered by gavage to male rats, 5 days per week, for 103 weeks. Doses of 0, 25, 50, or 100 mg/kg benzene in corn oil were administered by gavage to female rats and to male and female mice for 103 weeks. Ten animals in each of the 16 groups were killed at 12 months, and necropsies were performed. Hematologic profiles were performed at 3-month intervals. For the 2-year studies, mean body weights of the top dose groups of male rats and of both sexes of mice were lower than those of the controls. Survivals of the top dose group of rats and mice of each sex were reduced; however, at week 92 for rats and week 91 for mice, survival was greater than 60% in all groups; most of the dosed animals that died before week 103 had neoplasia. Compound-related nonneoplastic or neoplastic effects on the hematopoietic system, Zymbal gland, forestomach, and adrenal gland were found both for rats and mice. Further, the oral cavity was affected in rats, and the lung, liver, Harderian gland, preputial gland, ovary, and mammary gland were affected in mice. Under the conditions of these 2-year gavage studies, there was clear evidence of carcinogenicity of benzene in male F344/N rats, female F344/N rats, male B6C3F1 mice, and female B6C3F1 mice. In male rats, benzene caused increased incidences of Zymbal gland carcinomas, squamous cell papillomas and squamous cell carcinomas of the oral cavity, and squamous cell papillomas and squamous cell carcinomas of the skin. In female rats, benzene caused increased incidences of Zymbal gland carcinomas and squamous cell papillomas and squamous cell carcinomas of the oral cavity.(ABSTRACT TRUNCATED AT 400 WORDS)

Sources of variability in rodent carcinogenicity studies

A number of factors may influence tumor rates in rodent carcinogenicity studies, including the animal room environment, genetic differences, food consumption/weight gain, survival/age of the animals, identification of gross lesions, pathology sampling procedures and preparation of the histology slides, and histopathologic diagnosis. The relative importance of these factors is evaluated, making use of laboratory animal carcinogenicity data from the National Toxicology Program and from other sources. An investigator must be aware of these potentially confounding factors, so that appropriate measures can be taken to reduce or eliminate their impact on the interpretation of study results. Certain potential sources of within-study variability can be controlled by appropriate experimental design and by proper conduct according to standard operating procedures. The effect of certain factors influencing tumor prevalence may be magnified when variability from study to study is considered, and thus it may be difficult to formulate a biologically meaningful statistical analysis that uses historical control data in a formal testing framework.

Sources of Variability in Rodent Carcinogenicity Studies

Sources of Variability in Rodent Carcinogenicity Studies. HASEMAN, J. E., HUFF, J. E., RAO, G. K, AND EUSTIS, S. L. (1989). Fundam. Appl Toxicol. 12, 793–804. A number of factors may influence tumor rates in rodent carcinogenicity studies, including the animal room environment, genetic differences, food consumption/weight gain, survival/age of the animals, identification of gross lesions, pathology sampling procedures and preparation of the histology slides, and histopathologic diagnosis The relative importance of these factors is evaluated, making use of laboratory animal carcinogenicity data from the National Toxicology Program and from other sources. An investigator must be aware of these potentially confounding factors, so that appropriate measures can be taken to reduce or eliminate their impact on the interpretation of study results. Certain potential sources of within-study variability can be controlled by appropriate experimental design and by proper conduct according to standard operating procedures. The effect of certain factor influencing tumor prevalence may be magnified when variability from study to study is considered, and thus it may be difficult to formulate a biologically meaningful statistical analysis that uses historical control data in a formal testing framework.

Do cage effects influence tumor incidence? An examination of laboratory animal carcinogenicity studies utilizing Fischer 344 rats.

Approximately 125 carcinogenicity studies in Fischer 344 rats conducted by the National Toxicology Program (NTP) were examined to determine the frequency with which cage effects were associated with observed carcinogenic responses. All studies involving groups of 50 rats housed five per cage and showing evidence of chemically-related carcinogenicity were considered. For each of these experiments, two statistical analyses were carried out for each dosed and control group: (i) a test to determine whether or not the occurrence of tumors clustered within cages; and (ii) an evaluation to determine whether or not tumor incidences differed significantly between differing cage shelf levels. These analyses showed that the numbers of statistically significant (P less than 0.05 or P less than 0.01) effects were consistent with the number expected by chance alone. Thus, cage-related factors appeared to have little or no impact upon tumor incidence in these particular studies. Experimental design protocols now used by the NTP (which include random assignment of animals to cages; random assignment of columns of cages to dosed and control groups; and periodic rotation of cage location) further reduce the likelihood that factors associated with the housing of the animals could influence tumor incidence in current studies.

Tobacco-specific nitrosamines, an important group of carcinogens in tobacco and tobacco smoke.

Tobacco-specific nitrosamines are a group of carcinogens that are present in tobacco and tobacco smoke. They are formed from nicotine and related tobacco alkaloids. Two of the nicotine-derived nitrosamines, NNK and NNN, are strong carcinogens in laboratory animals. They can induce tumors both locally and systemically. The induction of oral cavity tumors by a mixture of NNK and NNN, and the organospecificity of NNK for the lung are particularly noteworthy. The amounts of NNK and NNN in tobacco and tobacco smoke are high enough that their total estimated doses to long-term snuff-dippers or smokers are similar in magnitude to the total doses required to produce cancer in laboratory animals. These exposures thus represent an unacceptable risk to tobacco consumers, and possibly to non-smokers exposed for years to environmental tobacco smoke. The permission of such high levels of carcinogens in consumer products used by millions of people represents a major legislative failure. Indeed, the levels of tobacco-specific nitrosamines in tobacco are thousands of times higher than the amounts of other nitrosamines in consumer products that are regulated by government authorities. Although the role of tobacco-specific nitrosamines as causative factors in tobacco-related human cancers cannot be assessed with certainty because of the complexity of tobacco and tobacco smoke, several lines of evidence strongly indicate that they have a major role, especially in the causation of oral cancer in snuff-dippers. Epidemiologic studies have demonstrated that snuff-dipping causes oral cancer. NNK and NNN are quantitatively the most prevalent known carcinogens in snuff, and they induce oral tumors when applied to the rat oral cavity. A role for NNK in the induction of lung cancer by tobacco smoke is likely because of its organospecificity for the lung. Tobacco-specific nitrosamines may also be involved in the etiology of tobacco-related cancers of the esophagus, nasal cavity, and pancreas. Because they are derived from nicotine, and therefore should be associated only with tobacco, tobacco smoke and other nicotine-containing products, tobacco-specific nitrosamines as well as their metabolites and macromolecular adducts should be ideal markers for assessing human exposure to, and metabolic activation of, tobacco smoke carcinogens. Ongoing research has demonstrated the formation of globin and DNA adducts of NNK and NNN in experimental animals. Sensitive methods for the detection and quantitation of these adducts in humans would provide an approach to assessing individual risk for tobacco-related cancers.(ABSTRACT TRUNCATED AT 400 WORDS)

Use of dual control groups to estimate false positive rates in laboratory animal carcinogenicity studies

Tumor incidence data from 18 recently completed carcinogenicity studies utilizing male and female mice and rats were examined to determine if the frequency of significant ( p < 0.05) pairwise differences between the two concurrent control groups employed in these experiments exceeded chance expectation. Although marked study-to-study variability was observed for some tumors, no evidence of extra-binomial within-study variability between the two concurrent control groups was found. The total number of observed significant ( p < 0.05) paired-control differences was virtually identical to what would be expected from the usual binomial model assumptions; the corresponding overall observed (44%) and expected (47–50%) false positive rates were essentially the same. While one should not overgeneralize the implications of these findings, these results should lessen concerns that elevated false positive rates resulting from extra-binomial within-study variability might be adversely affecting the interpretation of long-term laboratory animal carcinogenicity studies. On the other hand, these results reaffirm the conclusions of other investigators that (particularly for commonly occurring tumors) more stringent evidence than an isolated p < 0.05 effect should be required before an increased tumor incidence is regarded as biologically significant; otherwise, the study may have an unacceptably high false positive rate.

Use of Dual Control Groups to Estimate False Positive Rates in Laboratory Animal Carcinogenicity Studies

Use of Dual Control Groups to Estimate False Positive Rates in Laboratory Animal Carcinogenicity Studies. HASEMAN, J.K., WINBUSH, J.S., AND O'DONNELL, M.W., JR. (1986). Fun-dam. Appl. Toxicol. 7, 573-584. Tumor incidence data from 18 recently completed carcinogenicity studies utilizing male and female mice and rats were examined to determine if the frequency of significant (p < 0.05) pairwise differences between the two concurrent control groups employed in these experiments exceeded chance expectation. Although marked study-to-study variability was observed for some tumors, no evidence of extra-binomial within-study variability between the two concurrent control groups was found. The total number of observed significant (p < 0.05) paired-control differences was virtually identical to what would be expected from the usual binomial model assumptions; the corresponding overall observed (44%) and expected (47-50%) false positive rates were essentially the same. While one should not overgeneralize the implications of these findings, these results should lessen concerns that elevated false positive rates resulting from extra-binomial within-study variability might be adversely affecting the interpretation of long-term laboratory animal carcinogenicity studies. On the other hand, these results reaffirm the conclusions of other investigators that (particularly for commonly occurring tumors) more stringent evidence than an isolated p < 0.05 effect should be required before an increased tumor incidence is regarded as biologically significant; otherwise, the study may have an unacceptably high false positive rate.

A Semiparametric Model for Regression Analysis of Interval-Censored Failure Time Data

Left-, right-, and interval-censored response time data arise in a variety of settings, including the analyses of data from laboratory animal carcinogenicity experiments, clinical trials, and longitudinal studies. For such incomplete data, the usual regression techniques such as the Cox (1972, Journal of the Royal Statistical Society, Series B 34, 187-220) proportional hazards model are inapplicable. In this paper, we present a method for regression analysis which accommodates interval-censored data. We present applications of this methodology to data sets from a study of breast cancer patients who were followed for cosmetic response to therapy, a small animal tumorigenicity study, and a clinical trial.

Cancer risk from the lifetime intake of Ra and U isotopes.

From extensive human data on the induction of skeletal cancers (bone sarcomas and carcinomas of the head sinuses) by 226Ra, 228Ra and 224Ra, the cumulative lifetime risk to 1 million people, each ingesting 5 pCi of a Ra isotope per day, was calculated to be nine bone sarcomas plus 12 head carcinomas for 226Ra, 22 bone sarcomas for 228Ra, and 1.6 bone sarcomas for 224Ra. Assuming that the risk per rad of average skeletal dose is equal for 226Ra and the U isotopes with half-lives exceeding 1000 yr and that the equilibrium skeletal content is 25 times the daily ingestion of 226Ra, but 11 times the daily ingestion of long-lived U, the cumulative life-span risk to 1 million persons, each ingesting 5 pCi per day of 233U, 234U, 235U, 236U or 238U, is estimated to be about 1.5 bone sarcomas. The U risk is not well established and additional research is needed on the metabolism of U in humans and its carcinogenicity in laboratory animals. These estimates assume linear dose responses. However, if incidence varies with the square of dose, virtually no induced cancers would be expected from these levels of radioactivity.

Cancer Mortality Among Patients With Hansen's Disease

For the evaluation of cancer risks associated with immunodeficiencies experienced by patients with Hansen's disease (leprosy) and for the assessment of possible adverse effects of dapsone therapy, a follow-up study was conducted of 1,678 patients admitted to the National Hansen's Disease Center in Carville, La., between 1939 and 1977. Overall, no substantial cancer mortality was observed (standardized mortality ratio = 1.3), nor was there any excess among patients exhibiting defects in cellular immunity by virtue of lepromatous forms of the disease. Notable was the absence of any significant excess of lymphoma (5 observed vs. 2.3 expected), despite the predominance of this tumor in certain other immunodeficiency states. Several cancer sites (oral, bladder, and kidney) occurred excessively, but reasons for the elevations were obscure. Although dapsone has been implicated as a carcinogen in laboratory animals, the use of sulfones, including dapsone, did not appear to affect significantly the risk of any cancers in this population.

Metabolism of tobacco-specific N-nitrosamines by cultured human tissues.

N'-Nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) are present in cigarette smoke and snuff and are carcinogens in laboratory animals. In tobacco smokers, the buccal mucosa, trachea, esophagus, bronchi, and peripheral lung are exposed to smoke containing significant amounts of these N-nitrosamines. The results of the present study demonstrate that explants of these tissues as well as of the urinary bladder have the capacity to metabolize NNN and NNK by alpha-carbon hydroxylation. This metabolic pathway yields alkyldiazohydroxides, which are reactive and DNA-damaging electrophiles. The extent of alpha-carbon hydroxylation of NNN and NNK in human tissues was only 1/10th to 1/100th of that in animal tissues. Although the levels of alpha-carbon hydroxylation of NNN among different tissues of the same individual were similar, a 10-fold variation among individuals was observed. Reduction of the NNK carbonyl group was a major metabolic pathway observed with all human explants and may occur in the surface epithelia of the respiratory tract of smokers. These results provide further evidence that tobacco-specific N-nitrosamines could play a role in cancers related to the smoking and chewing of tobacco.

Benzidine dihydrochloride: toxicological assessment in mice during chronic exposures.

Although benzidine is recognized as a bladder carcinogen in humans and a liver carcinogen in laboratory animals, its toxicological effects appear to be extended to several other endpoints. This economically important chemical is the base for over 200 dyes and is used extensively in manufacturing. In a chronic lifespan study lasting 33 months, both sexes of F1 hybrid (genetically homogeneous) and monohybrid cross (genetically heterogeneous) mice from BALB/c male and C57BL/6 female crosses were exposed to benzidine dihydrochloride in their drinking water at concentrations of 0, 20, 30, 40, 60, 80, and 120 ppm for the females, and 0, 30, 40, 60, 80, 120, and 160 ppm for males. Animals were removed from the study when they were dead or moribund. In addition to hepatocellular carcinomas, there were several other toxicological end-points identified that appeared to be related to the administration of benzidine. Dose-response trends were noted for pigmentation of the spleen, hepatic cytological alterations, hyperplasia of the bile ducts, megakaryocytosis of the bone marrow, vacuolization of the brain, adenoma of the Harderian gland, atrophy of the ovaries, and angioma of the uterus. Also, dose-related effects were noted with respect to time to lung tumor and time to mortality due to reticulum-cell sarcomas.

Epidemiologic approaches to cancer etiology.

Epidemiology has contributed substantially to knowledge about the causes of human cancer and provides the basis for preventive measures. The ap­ proach dates from 1700 when the occupational physician Bernadino Ramazzini reported that nuns were at high risk of breast cancer, and 1775 when the surgeon Percivall Pott found that chimney sweeps exposed to soot were prone to scrotal cancer (1). In more recent times the initial leads to epidemiologic investigations have continued to come from astute clinicians who noted an excessive number of patients with the same tumor and traced the cluster to a particular cultural, occupational, or iatrogenic exposure. New insights into cancer etiology are provided also by experimental ap­ proaches to detect carcinogens in laboratory animals or mutagens in short­ term test assays, and to clarify basic mechanisms of carcinogenesis. In recent years the pace of epidemiologic and experimental research in cancer etiology has accelerated, including efforts to identify environmental deter­ minants, which are generally held responsible for a large proportion of cancers in the general population (2).

Nitrata and Human Cancer: A Review of the Evidence

Nitrites, derived mainly from ingested nitrates, may react in vivo with secondary nitrogen compounds occurring naturally in certain foods to form N-nitroso compounds, which are potent carcinogens in laboratory animals. This paper reviews the mechanism of their formation and the evidence from epidemiological studies that high nitrate ingestion is involved in the aetiology of human cancer.

Erratum: Pesticides: Three EPA Attorneys Quit and Hoist a Warning Flag

The article, "Pesticides: Three EPA attorneys quit and hoist a warning flag" (19 March) referred to aldrin and dieldrin and heptachlor and chlordane as "two pairs of compounds found to be potent carcinogens." Most uses of both aldrin/dieldrin and heptachlor/chlordane were in fact ordered suspended by the administrator of the Environmental Protection Agency as an "imminent hazard" to human health and the environment. But some readers may take the words "potent carcinogen" to mean that a compound has been determined by federal authorities to be carcinogenic in laboratory animals at relatively low dose levels. No such finding has been made with respect to heptachlor/ chlordane. In the case of these compounds, the administrator overruled the decision of the administrative law judge, who held that to suspend their use as an imminent hazard was not justified. He felt "hesitantly unwilling at this time to find that heptachlor/chlordane are conclusively carcinogens in laboratory animals." The administrator concluded, however, that heptachlor is a carcinogen in both the rat and the mouse. Weighing the risks of continued use of heptachlor/chlordane against the benefits, he decided that most uses should be suspended. His ruling is now under review by the U.S. Circuit Court of Appeals for the District of Columbia.-L.J.C..

Pigmentary degeneration of the retina induced by N-methyl-N-nitrosourea. An experimental study in syrian hamsters.

N-methyl-N-nitrosourea (NMU), a potent carcinogen in laboratory animals, has been shown to have a retinotoxic effect in Syrian hamsters. Six animals received two 5-mg doses of NMU intravenously at weekly intervals and six other animals received a single 5-mg dose. The retinal changes were fairly uniform and bilateral. The earliest lesion, observed at seven days consisted of arcades confined to the outer nuclear layer. The pigment epithelium revealed degenerative changes. Later there was widespread destruction of the rods and outer nuclear layer. The end stage consisted of a thin remnant of retina with an attenuated inner nuclear layer in direct contact with the choroid. There was intraretinal migration of pigment. The optic nerve and the anterior ocular structures including the lens showed no abnormality.