Document gDRqX4kbr9nqRvk7L6vopqZJJ

By Joseph C. Arcos A !, f * ,* T t aily we are exposed to hundreds of chem stated that approximately only 350 chemical com Dical agents in an infinite number of combina tions, by way of the air, the drinking water, pounds were then on long-term tests in laboratory animals. The performance of these bioassays re the food, by skin contact, occasionally by way oqfuires an average of 3 yr. The National Cancer In medication, and in industrial working environ stitute and its contractual-testing laboratory net ments. A number of these agents have the ability to work represents by far the largest organization of induce Ij* '`pr-'Cf u. . "* r iU*.* , r#r ** * - c , i ~ v 14 . %" H H- + tf l- ^____________ j, this type in the world. At present the total world ca pability of testing chemicals for carcinogenic activ 4f- a, > ^ JMT - -L*^ j:i- ity is at most twice that of the National Cancer In H*p ** <- *-' .l,. , * . - ' ' * -j:, < ! f -j * \ V VHf . It'.--'"' ^ -.1- 1 - , ^ ,_Uw J - -"l-i* *'.%-! ` * ' * ^ ' # iJn- Jfc : . .., H > tarf* .'* ** '<* -K^v * . ! * 1-int stitute's organization. Hence, it is probable that a minimum of 300 new and untested or inadequately tested organic chemicals find their way yearly into consumer items or industrial use, world-wide. Some The aim of this article is to provide a general of the environmental pollutants are stable chem background for nonspecialist readers of this jour ically and are biodegraded very slowly. Moreover, nal in the light of which they may meaningfully cor this already pessimistic picture does not take into relate and to some extent critically evaluate the sig account the melancholy fact that a considerable nificance of scattered information obtained on \ number of chemicals have been put to use in highly cancer-inducing chemical agents. The article de industrialized countries before their present strin scribes the methods of testing chemicals for car- >) gent criteria of licensing for consumer use were cinogenic activity, the guidelines for evaluating the $ established. km significance of the test results, the major classes of chemical carcinogens and their mechanism of bio- ; h logical action, and the types of interaction that oc- < Testing procedures * . Ii>l cur when several carcinogens act in combination. Bioassays J, Since humans are mammals, chemical agents are Scope of the problem usually assayed in mammalian species. Small fi Close to 200,000 new organic compounds are synthesized throughout the world yearly, and it is conservatively estimated that well over 1000 of these will eventually be put to some use, and, thus, into the environment. Hence it is necessary to test chemicals for carcinogenic activity before they come in contact with human populations. Yet, the "Carcinogenesis Technical Report** No. 1, released in May, 1976 by the National Cancer Institute, rodents (rats, mice, and hamsters) are generally used. This is because of the statistical necessity of using large groups, and because short-life-span spe cies (average 2-3 yr) are preferred for reasons that will be discussed below. While a great variety of ways of administration have been tried, substances are most commonly assayed by: 1. Feeding; mixing the substance into the diet, dissolving it in the drinking water, or administering it directly into the stomach by special tube. i^ JrT-*: 2. Skin "painting," where small volumes of a Ij* I -4 solution of the substance in a solvent are deposited Dr. Areas is Professor, Tulane Medical Center, and CoDirector, Biochemistry Section, U.S. Public Health Service Hos pital, New Orleans. He was involvedfor the past 25 years in the study of chemical carcinogenesis and is the author of over 120 scientific articles and three books on the chemical induction of on the shaved surface of the skin. The term painting originated from the fact that in the early years of such assays (around 1940) the solution of the sub stance tested was actually applied to the skin with a cancer. small brush. ! \ T * % : I I VAB.0001034620 , AMERICAN LABORATORY : 65 i I PRESENTING...... SUPEROXTM..... A NEW GCPHASE SUPEROX is a new; specially prepared, high quality polar stationary phase for use in gas chromatography. SUPEROX is available in two molecular weights. These are 600,000 (SUPEROX 0.6) and 4,000,000 (SUPEROX 4). SUPEROX exhibits the typical undefined melting point of high molecular weight polyoxirans and has a lower temperature limit of 65C. If oxygen is completely avoided in the carrier gas stream, SUPEROX can be used at 300C and even as high as 325C for brief periods. SUPEROX does not contract into droplets on bare glass surfaces which gives it a great potential for use in glass capillary columns SUPEROX 4 SQUALANE/SQUALENE Column: 1.5m x 3mn\ 3% SUPEROX 4, Chromoaorb W-HP, 80/100 mmh aor, Omdor Temp: 3N Column Tamp: 300"C Flowrate: 23ml H2 / minute Sampla: Mixture of Squalarva 8 Squalana FOR MORE IMFORMA T/ON WRITE FOR BULLETIN 5330-32 TO ORDER SUPEROX SPECIFY: 5330 5332 SUPEROX 0.6 SUPEROX 4 $18.00/1 Og 18.00/10g 1 SOLVENT 2 SQUALAN! 3 SOUALENE Z2i Tint. SmnA MVIII0JP" Trmdamtrk 0/ AHt*ch Me. (pvftrauiit 1 \i 20 CANCER continued Circle Reader Service Card No. 100 2670 3. Subcutaneous injection of the substance or its solution once or at repeated intervals. 4. Inhalation of fine suspensions (aerosols) of the substance or introduction of its solution into the trachea by means of a special tube (intratracheal in tubation). When a bioassay is carried out for reasons of public health considerations of human safety (rather than as an experiment to explore a scientific problem, such as biochemical studies or structureactivity relationships), the route of administration of the chemical mimics human exposure whenever possible. At the end of the testing period the surviving ani mals are killed, completely autopsied, and every ab normality is diagnosed by histopathological ex amination of tissue samples. The tissue samples are "Fixed" (to forestall deterioration), stained with an appropriate dye or dyes to render them observable and provide contrast to microscopically visible tis sue structure, and then are submitted to histopathology. This examination will reveal any change (resulting from the administration of the test com pound) in the characteristic normal microscopic pattern of the particular tissue at hand, in the size and shape of the cells, the appearance and arrange ment of intracellular components, etc. The totality 66 : JUNE 1978 of this information enables the pathologist to diag nose whether or not the tissue has undergone a neo plastic change, to determine the cell origin (i.e., histological type) of any tumor, and to estimate the degree of its malignancy. Two important parameters are obtained in a bio assay experiment. These are a) the tumor incidence, representing the percentage of animals bearing at least one tumor in the animal group considered (for example, 40 tumor-bearing animals in a test group of 100 would represent a tumor incidence of 40%); and b) the latent period, which is most commonly considered to be the number of days from the be ginning of administration of the compound until the first tumor in the group is observed. To characterize the carcinogenic potency of a chemical agent by one number, several carcino genicity indexes have been proposed and used. These indexes are calculated from the tumor inci dence and latent period, using various formulas. One of the most widely used is the (ball index: Iball index =, tumor incidence <<?.) x |00 latent period (days) This index is based on the logic that the higher the tumor incidence, and the shorter the latent period (that is, the smaller the number of days) necessary VAB.0001034621 Truly multi-technique surface analysis A Dual-Anode XPS Monochromatic UPS d .it A BtNDtNQ ENERGY For Literature Circle No. 95 SIMS *1 ~ T-TM ' 'JT' *ir - . J'iX' , *- > y - - * -1 * ,p . ~ m1lFKFKInf, *,*4'..-**,,v-r*.V$>.:.wi.&s*ff^-r4t..?.. . -! * fi ' ,1 . ? . - 4. __ 1 < ^ .j n * + * *V 'i V L. H^ t-P The ES300/ DS300 Electron Spectrometer /Data System pro vides true multi-technique surface analysis, not only in the range of excitation sources, but also in the types and shapes of samples, the sample handling facilities, the modes of scanning and the data handling facilities. In a nutshell, herefs the ES300/DS300 picture. m pow<jcrSj pellets, strips, blocks... accommodated Solids, gases, liquids (in condensed state) Rough samples with no sensitivity loss No critical sample positioning required Large, multi-port sample preparation chamber Fast insertion airlock--60 sec insertion Highest signal/background available Highest resolution on the market (see inset) Highest signal/background available (see inset) Foreground /background data system Real U.H.V. system--10-10 torr guaranteed -. KRATOS, INC. . AEI Scientific Instruments 8322 Clairemont Mesa Blvd. San Diego, CA 92111 Telephone (714) 292-0925 Ir I for inducing the first tumor, the more potent the compound that has been tested. The final results of bioassays are considered in the light of the following five principles. 1. A positive result, i.e., a chemical agent is found to be carcinogenic, carries a much greater weight than a negative result. This is because it can never be excluded that a compound, found inactive under certain experimental conditions, may prove to be carcinogenic when tested in another species by different routes and ways of administration, and under different dietary conditions. 2. It is a general consensus among investigators of chemical carcinogenesis that for any testing experiment to be meaningful at all, whether carried out for reasons of public health safety considerations or as a purely scientific study, it must be carried out for a minimum of 1 yr, unless a statistically significant tumor incidence would happen to become manifest earlier. 3. No chemical compound may be stated safely to be devoid of carcinogenic activity toward man unless it has been found inactive when tested in several mammalian species and by several routes of administration for a length of time corresponding to one half (or even better, the whole) life span of each * species. This statement is based on these frequent findings: A carcinogen that is inactive in one species may be highly active in another; the susceptibility of a species to a given carcinogen also depends on the genetic strain, sex, and dietary conditions; the de tectability of carcinogenic activity often depends on the route of administration. Moreover, it is well established that the incidence of cancer in humans increases with the age group, and that the tumor, if its origin can be traced to a single or repeated expo sures to a carcinogenic agent, will often appear only decades after exposure. Hence, the preference for ' using short-lived test species and to test for the en- ; tire life span can be understood. ;j 4. Some considerations of experimental cancer ^ research may not be carried over to public health j considerations of human safety. A chemical agent < that would induce a 1% net tumor incidence over that of a control group, even though .statistically significant. Would not be regarded as really mean- I! j j ingfully carcinogenic from the standpoint of lab- I oratory experimentation. It is evident, however, that a chemical agent would Jt>e regarded as a major health hazard if it were known with certainty to produce 1,000 cancer patients per 100,000 popula- tion. 5. The latter considerations lead us to examine the statistical limitation of any bioassay procedure. To illustrate the point, consider that a compound is VAB.0001034622 AMERICAN LABORATORY : 67 A # sorbed and disappears if carcinogen application is discontinued at this point. However, if the applica tion is continued for 11 weeks, the tumor turns ma lignant (Figure 2b) and will continue to grow and ultimately will kill the animal. Once the tumor has turned malignant the process is irreversible. (Histopathological examination has shown that the tumor p in Figure 2b was a keratinizing epithelioma). While hydrocarbons have a predominantly local effect at the site of application, such as the mouse skin tumor shown, oral administration of hydrocar bons produces tumors in distant tissue targets, mainly malignant mammary and lung tumors, and leukemia. The reason why hydrocarbon carcinogens, and especially benzo[a]pyrene, are so ubiquitous is that they readily arise from almost any kind of organic material by pyrolysis. Thus, fe&izpfglpyrenf has been identified in food materials and in consumer and industrial products, the processing of which in volves high temperatiires aJbe^'fncInd^Tdrirxafiv^ Figure 2 Two stages in the development of a skin tumor induced in the mouse using benzofajpyrene. lution of lakes and rivers with polycyclic hydrocar bons originates mainly from the untreated effluents rings, a considerable variety of structures exists. of industrial plants and from the lubricating oils V Benzo[a]pyrene (also known as 3,4-benzopyrene), and exhaust fumes of ships. Some of these pollu was the first hydrocarbon identified to be a strong tants eventually find their way into the water table carcinogen. It was isolated from coal tar, and the and, hence, into the drinking water. lead that pointed to this source was the unusually Figure 3 summarizes the chemical mechanism of high skin cancer incidence in workers in the indus the formation of benzo[a]pyrene from organic tries using coal tar. 3-Methylcholanthrene is an other strong carcinogen; it has been obtained by synthesis and it is not present in tar. Benz[a]anthracene (also known as 1,2-benzanthracene) and di- ORGANIC MATERIALS Pfroljtii (Fossil fuels, foodmoterials, refuse, etc I aliphatic fragments benz[a,h]anthracene (also known as 1,2,5,6-di^ benzanthracene) do occur in tar; however, they are weaker carcinogens than benzo[a]pyrene and arise during tar formation in lesser amounts. The four i compounds shown in Figure 1 are only representa tive examples; a great variety of hydrocarbon car cinogens are known. ti ! Benzola]pyrene is the most widely and ubiqui tously distributed hydrocarbon carcinogen in the environment. It is also the main hydrocarbon car cinogen in cigarette smoke. Figure 2 exemplifies the i result of application of this compound on the mouse skin. The mouse shown in Figure 2a re Benzo[i]fiuorantbene i ceived, twice weekly, applications of 0.3 mg of benzo[a]pyrene for 7 weeks. The tumor seen is a papilloma, a benign tumor, which most often is re- Figure 3 Molecular mechanism of the formation of benzofajpyrene and benzo[j]fiuorathene from lower hydrocar bons during pyrolysis. VAB.0001034623 AMERICAN LABORATORY : 69 HI T i 4-Dimethylaminoslilbene 4-Dimelhyloijiinoazobenzene (Butler Yellow) 2-Naphthytamine <9 4S 2-Aminofluorene CHj-CH^CI CHjrCHj--Cl Mustard Gas /Hz* \ CHj-CI bis-chloromethyhether 0 Dioxane HiC-CH-CH-CH2 'V V Diepoxytw*one ? CI-C-CI Cl Carbonletrochloride HjN-COO-CjHj Ethylcarbomote (" urethane") A Propiolactone H5Cz~N"H-CjH9 Azoethane CHi-CH-CI Vinyl Chloride Figure 4 Varieties of chemical carcinogens. materials during pyrolysis. A common pyrolytic de composition product of all organic compounds is acetylene. However, molecules of acetylene in turn undergo gradual recombination at high tempera tures to yield larger and larger structures, ultimately yielding polycyclic hydrocarbons. These com pounds are relatively stable at high temperatures and, hence, accumulate: as pyrolysis progresses. One of these hydrocarbons is benzo[a]pyrene. As stated earlier, chemical carcinogens represent a considerable variety of structures. Figure 4 shows several structural types other than polycyclic hydro carbons. The histories of these illustrate further the insidious hazard that chemical carcinogens repre sent, as well as their ubiquitous presence. Aromatic amines These have the general structure H2N-aryl or (alkyl)2N-aryl; attachment of an amino or dimethylamino grouping to an otherwise inactive, lowmolecular-weight aryl moiety often confers power ful carcinogenic activity. 2-Naphthylamine and benzidine were extensively used before 1960 as intermediates in the manufacture of certain textile dyes. They are powerful bladder carcinogens to man and animals, and account for the fact that dye factory workers have a bladder cancer risk 200-300 times higher than the population at large. Manufac ture of these compounds is now tightly restricted. 2-Aminofluorene (as N-acetyl) was proposed for use as an insecticide in the early 1940's. It is indeed an excellent insecticide. However, virtually by acci dent it was discovered, fortunately before it was re leased for use, that it is a very powerful carcinogen toward most abdominal organs, the ear duct, and the mammary gland in rodents. 4-Dimethylaminoazobenzene, as its alternate name "Butter Yellow" indicates, was used to color butter and cooking oils up to about 1935, when it was discovered to be a powerful carcinogen toward 70 : JUNE 1978 the liver and to some extent toward the skin in ro dents. It is still in use as an industrial solvent dye. A great variety of azo dyes has been found to be car cinogenic. Many of the "certified food colors" are azo dyes, the relative safety of which was estab lished before licensing. A liphatic carcinogens These are by themselves an extremely structurally heterogeneous group; their only common denomi nator is that they are chemically reactive as such or are transformed to chemically reactive agents (proximate carcinogens) by metabolism (see Part 11 of this article), and they interact with nucleic acids and proteins in the target tissue to bring about the change to malignancy. For example, /3,/3-dichlorodiethyl sulfide or sulfur mustard, as the compound is commonly known, was introduced in World War I by the German military high command as a potent vesicant warfare agent at the Belgian front, at Ypern, against the French troops. A number of the infantrymen who survived the gassing developed malignant lung tumors decades later. Subsequently, the compound was shown to be carcinogenic also in rodents. Carcinogenicity is maintained if the sulfur atom is replaced by an --NH-- group (nitrogen mustard). Various compounds in which a nitrogen mustard group is linked to another moiety were found to be carcinogenic. Dioxane is a common solvent used in tank-car quantities in the chemical industry. It is extensively employed in laboratories in scintillation counting and has many other uses. It is a weak to moderately active liver carcinogen, active also to some extent toward the kidne$ and lung and toward the epitheli- m um of the nasal passage in rodents. Carbon tetrachloride, a medium-active liver car cinogen in mice and hamsters, was extensively used until not so long ago in the dry cleaning industry and was widely used in the chemical industry as an VAB.0001034624 CANCER continued Diolkyinitrosomines Al kylocylrntrosomincs A Alkylorylnitrosomirm >N-N=0 * Figure 5 Principal types of carcinogenic nitrosamines. CtHe C5H7 CHi HsCt-0-C-i 11 0 XXL extractive solvent. Its close chemical relative, chlo roform, is also carcinogenic. Chloroform was used several decades ago as a surgical hypnotic agent and until very recently as a component of cough syrups and ear drops. etc Ri^Ri mixed alkyl and more complex polor-nonpolar chains cyclohexyl H|C-C- II N*0 0 Cvcioofkvlnitrosomines 0 1I NO NO NO NO NO Urethane (also known as ethyl carbamate) was used medicinally as a hypnotic agent; it is still used in veterinary medicine. This compound produces l NO NO NO pulmonary tumors in mice, rats, and chickens; it also produces liver tumors in occasional animals. Vinyl chloride is the starting material in the man ufacture of the plastic polyvinyl chloride. It was compounds known collectively as nitrosamines. also used until recently as the propellant gas, or Figure 5 illustrates the principal classes of carcino component thereof, in spray cans. Recent industrial genic nitrosamines. The common denominator of statistics demonstrated that vinyl chloride is a liver these compounds is that they all bear a nitroso carcinogen to humans, and its carcinogenicity to group linked to a secondary nitrogen atom. Bioas ward this organ was confirmed also in laboratory say results with about 200 nitrosamines so far sug animals. gest that probably as much as 90% of all hypothet The insecticides aldrin, endrin, dieldrin, mirex, ically possible nitrosamines will prove to be carcino and DDT, the herbicide atrazine, and the polychlo genic. The most common and widely distributed ni- rinated biphenyls (PCB's) used as plasticizer addi trosamine is dimethylnitrosamine. The primary tives (not shown in the figure), also belong in this tissue target of the carcinogenic action of dimethyl structurally miscellaneous group. nitrosamine in rodents is the liver; the secondary targets are the kidney and lung. Figure 6 illustrates lymphatic tumors, and leukemia in occasion the carcinogenic action of dimethylnitrosamine to al animals. The PCB's are among the most persis ward the liver in the rat. Figure 6a shows the ex tent organic chemicals in the environment. posed abdominal organs of a normal rat. Figure 6b shows the abdominal organs of a rat that received Nitrosamines 0.4 mg of dimethylnitrosamine daily, 5 times weekly, for 28 weeks. Note the large hepatic tumor In terms of total impact as environmental hazard, masses, increasing the overall size of the liver close however, the chemical family of carcinogens most to fivefold. These tumors are malignant (overwhel important next to the hydrocarbons comprises the mingly hepatocellular carcinomas). Dimethylnitros amine was shown to be carcinogenic in all the 16 species in which it was tested so far. A B The nitrosamines are potent, multitarget carcino gens. Depending on the type of nitrosamine, the tissue targets attacked include the lung, liver, kid ney, bladder, pancreas, stomach, and different re gions of the nervous system; some induce leukemia. Nitrosamines are formed with extreme ease from any secondary amine and nitrous acid; for example. Figure 6 a) Abdominal organs of a normal rat and b) ab dominal organs of a rat that was administered dimethyl nitrosamine. In thfy carcinogen-treated animal, large liver tumor masses are seen. 72 : JUNE 1978 VAB.0001034625 A The new Perkin-Elmer Model 650-10 is a com pact medium-priced fluorescence spectropho tometer only 20" wide. But it performs like a big expensive instrument. Model 650-10 delivers the highest sensitivity for the dollar, thanks to highefficiency ruled concave gratings and a proprietary optical innovation. You can also measure emission bands close to the excita tion bands. The slit width is continuously adjustable down to 1.5 nm, another Perkin-Elmer exclusive that gives precision con trol for varying intensity and resolution. Up to now, only high-priced units offered this feature. There's also a large sam ple compartment that can be changed for specialized work, and a digital display of fluorescence intensity. These are some of the ad vantages that make the Model 650-10 easily the most advanced in its class. Get all the details on this bargain in fluorescence performance. Contact your Perkin-Elmer repre sentative or write PerkinElmer Corp., Main Ave.,, M.S. 12, Norwalk, CT 06856. Telephone: (203) 762-4639. PERKIN-ELMER Expanding the world of analytical chemistry. Circle Reader Service Card No. 66 ] the simple reaction origin of dimethylnitrosamine detected in the atmo sphere. (HjC)iNH + HONO-(H,C)2N--NO + H20 Consistent with the ease of formation, nitrosamines, and in particular dimethylnitrosamine, depicts the formation of dimethylnitrosamine. have been identified in a variety of consumer prod However, tertiary amines can also undergo nitrosa- ucts, e.g., processed fish, certain cheeses, cured tion following the general reaction: ham, and fried bacon. Moreover, most processed meat products contain sodium nitrite as an additive; r . processed meats and all fish also contain various R2N--CHR2 HQNCV secondary amines, sometimes at elevated levels. Nitrosamines that are already preformed are not usually found in commercially sold processed meat products. However, upon ingestion, owing to the low pH in the mammalian stomach, nitrous acid r2n--cr2 *-r2n will be liberated from the sodium nitrite and will in II turn rapidly react with the secondary amines also ON H ingested with the meat, to yield nitrosamines. It has + HjO been amply documented that in rodents administra - R2CO tion of a secondary amine together with sodium ni 4 R2N--NO MONO --r,NH trite is just as carcinogenic as the preformed nitrosamine. Part II of this article will discuss the naturally In the presence of nitrate-reducing bacteria, nitrates may serve as the source of nitrous acid. The nitrosa- tion reaction isxatalvzed thio- cyanateion, _______ here is evidence fy induced nitrosation of amines by nitrogen oxides and this may be the occurring carcinogens, the possible role of food for mulation and preparation practices, the role of , metabolism, the inorganic carcinogens, the cross reactions between carcinogenic effects, and the ex ternal and internal factors influencing carcinogen esis. Part II will also provide a bibliography for further reading. VAB.0001034626 AMERICAN LABORATORY : 73 I