Exp Toxic PathoI1992; 44: 481-487 Gustav Fischer Verlag Jena

Regulatory Pathology, American Health Foundation, New York, U.S.A.

Ac.celerated rodent bioassay predictive of chemical carcinogenesis MICHAEL J. IA TROPOULOS*) With 6 tables Received: September 31, 1992 Address for correspondence: MiCHAEL J. IATROPOULOS, M.D., Ph.D., Six Bruce Court, Suffern NY 10901, USA. Key words: Rodent bioassay; Carcinogenesis, chemical; Accelerated rodent bioassay; Chemical carcinogenesis, rodent bioassay

Introduction The overall cancer mortality rate was fairly stable between 1950 and 1990, increasing only by a modest 6% during this period. However, due to the decrease in mortality due to some other diseases, in 1990 cancer became the leading cause of death for women in the U. S. During the same period, there have peen major shifts in the occurrence of some forms of neoplasia. For example, there has been a substantial decline in Hodgkin's lymphoma, cervical and endometrial cancers, cancers of the stomach, rectum, urinary, bladder, thyroid, testis, pharynx and oral cavity. There has also been an increase in breast cancer, prostate cancer, colon cancer, melanoma, non-Hodgkin's lymphoma and multiple myeloma. For most neoplastic changes, increased age is associated with an increased incidence of the change. Other neoplastic diseases show a characteristic and different peak of incidence, such as the acute lymphoblastic leukemia of children, testicular neoplasia of young adults, and Hodgkin's lymphoma of middle age (17,21). Substantial progress has been made recently in the characterization of carcinogenesis. During carcinogenesis, the genetically programmed cell and tissue death is altered, disrupted and bypassed (9, 23). The restraining signals from the cell and tissue are subverted or ignored (1). The immunological surveillance is circumvented (19, 22). The need for growth factors from other cells is modified, leading to cellular isolation from paracrine influences (1, 5, 6). The cellular processes of differentiation and proliferation are irreversibly altered (3, 18). A new and more appropriate blood supply is created through angiogenesis (2). The amount of the causative agent or factor and the time for expression (long latency period) are the most important modifiers. From this perspective it becomes evident that the *) Secretary General, IFSTP; Head, Regulatory Pathology, American Health Foundation; Professor of Pathology, N. Y. Medical College; President, Labpath Management, Inc.

factors and/or agents causing neoplasia include: (a) genetic factors; (b) chemical agents (both genotoxic and agenotoxic); (c) physical agents (irradiation and foreign bodies); (d) viruses (DNA-Papilloma, Hepatitis Band RNAretro-viruses); and (e) a combination of these factors and agents (21, 24). Based on the above and using data from a Surveillance, Epidemiology and End Results program published by the NIH in 1990, we see the most common factor in carcinogenesis is the chemical agent (9). These agents come in the form of hormones, of the ingredients in tobacco, and of dietary ingredients (table 1). The most common sites in men are the lung, prostate, colon, urinary bladder, and hemopoietic/lymphatic systems, accounting for 72 % of all neoplasias. In women the most common sites are the breast, colon, lung, endometrium and hemopoietic/lymphatic systems, which together account for 71 % of all neoplasms (17). Utilizing the excellent IARC data monographs of 1987 (10), we see that, to date, 33 unique substances or processes, including irradiation, have been found conclusively to be carcinogenic to humans (table 2). Twenty-three of these 33 agents, or 70 %, are found to be conclusively DNA-reactive (20). Comparing the sites of neoplasia from the NIH (17) and the IARC data (10), we see that 3 out of 4 important sites in both genders from the NIH data are not even present in the !ARC data; namely colon, breast, and prostate, these being also the ones with the highest incidence. What is even more important, neoplasias at these sites are now increasing. Cancer of the lung is on the decline, because it can be prevented since the main causes of this neoplasia are already known. It is apparent from the information presented so far that the weight of evidence in human carcinogenesis is based mainly on epidemiological data (9, 14). Further complicating the situation in human carcinogenesis is the long latency period, and the fact that humans are exposed simultaneously Exp Toxic Pathol 44 (1992) 8

481

Table 1. Percent Incidence and Known Causes of the Most Common Neoplasms in Men and Women in the U.S.A. (1987). Site

Men

Women

Known Causes

Lung

20

11

Breast Prostate Colon and Rectum Urinary Bladder

29 14 10

*) Tobacco, Arsenics, Asbestos Bis(chloromethy)ether, Chromium Compounds, Mustard Gas, Radon Gas Ovarian Hormones Testosterone, Estrogen Animal Fat, Low Fiber Diet, Alcohol Tobacco, 4-Aminobiphenyl, Auramine, Benzidine, Chlomaphazine, Cyclophosphamide, Naphthylamine , Rubber Manufacturing Estrogen (Conjugated) HTLV-T, X-rays, HIY, Azathioprine, Benzene, Myleran, **) Comb. Chemo. , Chlorambucil, Melphalan Tobacco, Alcohol Tobacco UV-rays, Arsenics Ovulation Hormones Tobacco, Analgesics Salt, Tobacco

Endometrium Leukemia and Lymphoma 8 Oral Cavity Pancreas Melanoma Ovary Kidney Stomach

4 3 3 2

27 16 4

10 7 2 3 3 4 2

*) Main known causes are in bold face; **) Combined chemotherapeutic agents such als Mechlorethamine/Vincristine/

Procarbazine/Prednisone

to a variety of chemicals, some of which can either interact to increase the risk of neoplasia or interact to decrease the risk. From the non-occupational variables, the factors of lifestyle and dietary habits and genetic background, and also the lack of precise data op the level and duration of carcinogenic exposure, render the only approach purely empirical (9, 14, 23). In order to introduce a scientific approach to the issue of chemical carcinogenesis, chronic exposure of rodents to potential chemical carcinogens has been used for many years, in what is called the "Rodent Bioassay" (16). This bioassay has two consistent and pertinent comparative characteristics. The first is that the liver is the first-ranked site in the rat, although it is completely insignificant in human chemical carcinogenesis (8 , 17). The second is that hormone sensitive tissue sites rank very high in both man and rat (8, 10, 15). The advantages and disadvantages of the bioassay, which is still unvalidated , have been debated for many years with increasing intensity. In the present paper, we wish to introduce a scientific study protocol, addressing and correcting all the disadvantages of the rodent bioassay. This protocol is at the same time capable of appropriately extrapolating data from laboratory animal to man , thereby making a more accurate prediction of chemical carcinogenesis in man (11).

Experimental design and methods The experimental design of this specific "Accelerated Rodent Assay" (ARA) contains 20 groups utilizing 640 male and female rats (table 3LThis can be either expanded or reduced, based on the needs from the sub chronic and early chronic rat preclinical studies. The choice of rat strain should be based on existing toxicokinetic (histokinetic) and toxico482

Exp Toxic Pathol 44 (1992) 8

dynamic (xenodynamic) data , as well as dose-response and subchronic toxicity data (11). We recommend the rat because the rat is used in 99 % of safety assessment studies , even if the mouse is utilized later as part of the overall carcinogenicity assessment program. We recommend that the choice of organs and tissues of ARA be based on results obtained from subchronic (and chronic if available) toxicity studies. They should represent organs where appropriate, pertinent and consistent cyto- and histo-toxicity has been observed. The selection of tissue sites in the present generic protocol is based on organotropic carcinogenicity findings presented earlier (15). The initial cellular process which is modified during carcinogenesis is differentiation (11). Growth alone is not essential, because cancer cells divide even in the absence of growth (1). The types of growth are embryonal, fetal, neonatal , puberal growth and maturity. During the course of attaining maturity and differentiation , all cells of an organism grow through hyperplasia from clonogenic cells (0.1 % of the total population). Before they differentiate , these cells are called stem cells. They produce G j progeny cells . From the post embryonic cells, only the renewing type has this property for differentiation (table 4). At maturity, the cellular genome is fixed, i.e. attains strong DNA repression . In very few tissues from maturity on, some plUlipotential stem cells are in existence (7). From table 4 we see that differentiation and proliferation are mutually exclusive (7, 18). Therefore the effective stage of carcinogenicity testing should start with maturity , when differentiation is fixed. The rate of "spontaneous" mutations is also cell type dependent, which in tum depends on the level of cell differentiation. The higher this level, the lower the propensity of mutations coding for cell division, e.g., the static cells have a very low rate of spontaneous mutations (11). The type of mutations is also important. For example,

Table 2. Substances or Processes Associated with Neoplasia in Humans (1987) (Main known causes are in bold face.).

DNA Reactive

Substance/Process

Site

+ +

Aflatoxins 4-Aminobiphenyl Arsenics Asbestos Auramine Mfg. Azathioprine Benzene Benzidine Bis( chloromethy I )ether Boot and Shoe and Furniture Mfg. Chemotherapy for Lymphomas! Chlorambucil Chlornaphazine2 Chromium compounds, hexavalent Cyclophosphamide Diethylstilbestrol Estrogens (replacement therapy, steroidal, oral contraceptives sequential and combined3 ) Melphalan Methoxsalen (8-Methoxy Psoralen) plus UV-Rays Methyl-CCNU 4 Mustard Gas s Myleran 6 2-Naphthylamine Nickel Refining Phenacetin Radon Gas Rubber Mfg. Testosterone Tobacco (smoke, smokeless products, Betelquid) Treosulfan Vinyl Chloride UV-Rays X-Rays

Liver Urinary Bladder Lung, Trachea, Melanoma Lung, Pleura Urinary Bladder Lymphoma Leukemia Urinary Bladder Lung, Trachea Nasal Cavity Leukemia Leukemia Urinary Bladder Lung, Trachea Urinary Bladder Vagina, Cervix Endometrium

+ + + + + + +

+ + + + + + + +

+ +

+ + + + 1 2 3 4 5 6

=

= = = = =

Leukemia Skin Leukemia Lung, Trachea Leukemia Urinary Bladder Nasal Cavity Kidney Lung, Trachea Urinary Bladder Prostate Lung, Trachea, Urinary Bladder, Oral Cavity, Pancreas, Kidneys Leukemia Liver Melanoma Leukemia, Lymphoma

Mechlorethamine/Vincristine/Procarbazine/Prednisone N ,N-Bis(2-chloroethyl)-2-naphthyl-amine the combined have a protective effect against cancer of the ovary and endometriur 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea 1,1-Thiobis[2-Chloroethane]; bis(2-chlorethyl)sulfide l,4-Butanediol dimethane sulfonate

protooncogenes are activated by dominant mutations, whereas tumor suppressor genes are activated by recessive mutations (19, 22). Furthermore, the fidelity of DNA repair is also important (23). Whether this is also dependent on cell type and level of differentation is not yet known conclusively. What is known is that the overall DNA excision ' repair capacity in the rat is one fourth of that in man. It is also evident that the 3 major integrating systems, i.e., the endocrine, nervous and immune systems, are interdigitated and committed to a harmonious coordination of target sites they influence during adaptation. For example, inhibi-

tion of the cholinergic activity, especially the M3 receptor, induces hyperplasia. Further, 3 target sites, the hematopoietic system, uterus and pancreas, are under adrenergic trophic influence,. and not parasympathetic (11). In addition to the target sites, we routinely include as part of the microscopic evaluation the pituitary, thyroid, parathyroids, pancreas, adrenals, ovaries, testes, uterus, epididymides, seminal vesicles, urinary bladder and lungs. In this generic protocol, the liver of the mature rat has been selected as the first target site. The rat liver has some major physiologic differences from human liver (table 5). In Exp Toxic Pathol 44 (1992)

8

483

Table 3. Experimental Design. G. No. 1

2 3 4

5 6 7 8 9 10 11

12 13 14 15 16 17

18 19 20

Group IdentifIkation

Target Site

Weeks of Treatment 0, 10, 14, 26, 38

Diet Control Males Diet Control Females DEN Males DEN Females DEN Males + PB DEN Females + PB DEN Males + CpdX DEN Females + CpdX CpdX Males + PB CpdX Females + PB DEN Males + NTA CpdX Males + NTA DMBA Females DMBA Females + DES DMBA Females + CpdX CpdX Females + DES MNU Males MNU Males + BRA MNU Males + CpdX CpdX Males + BRA

Liver, Kidney Liver, Kidney Liver, Kidney Liver, Kidney Liver, Kidney Liver, Kidney Liver Liver Kidney Kidney Mammary Gland Mammary Gland Mammary Gland Mammary Gland NG Stomach NG Stomach NG Stomach NG Stomach

DEN 0-10 DEN 0-10 DEN 0-10; PB 14-38 DEN 0-10; PB 14-38 DEN 0-10; CpdX 14-38 DEN 0-10; CpdX 14-38 CpdX 0-14; PB 14-38 CpdX 0-14; PB 14-38 DEN 0-10; NTA 14-38 CpdX 0-14; NTA 14-38 DMBA 0-10 DMBA 0-10; DES 14-38 DMBA 0-10; CpdX 14-38 CpdX 0-14; DES 14-38 MNU 0-10 MNU 0-10; BRA 14-38 MNU 0-10; CpdX 14-38 CpdX 0-14; BRA 14-38

CpdX = The compound under investigation. Three doses per sex are recommended when given during weeks 14-38. When given during weeks 0-14, only one dose (the high dose) is necessary. Table 4. Cell patterns of Adaptation (A) and Cell Life Cycle (CLC)I) Duration. Cell Type

Cell Site

Patterns of A and CLC

Static

Neurons Striated-myocytes

Never undergo hyperplasia/entire lifetime

Terminal

Superficial g.i.t. epithelia, Keratinized epidermal cells, Superficial mucosal epithelia, PMN's, RBCs

Never undergo hyperplasia, destined for exfoliation/ individual time of exfoliation

Decaying

Ova

Never undergo hyperplasia, finite no. present at birth/sexually active time, then atrophy

Expanding

Chondrocytes, Osteocytes, Smooth and Cardiac Myocytes, Adipocytes, Fibrocytes

First hypertrophy then undergo hyperplasia when reversibly injured/third of lifetime

Intermediate

Endothelia, Mesothelia, Repatocytes

First hypertrophy, then undergo hyperplasia, especially when injured!2 to 7 days

Indeterminate Type 1

Brown adipocytes, Endocrinocytes

Some hypertrophy, some undergo hyperplasia when signaled/2 to 14 days

Indeterminate Type 2

Exocrinocytes

All undergo hyperplasia, esp. when surroundings are injured/2 to 21 days

Renewing Type 1

Thymocytes 2), Bone marrow cells

All undergo hyperplasia, esp. when organism is injured!24 hrs

Renewing Type 2

Epidermocytes, Respiratory duct cells, Endocrine duct cells, Exocrine duct cells, Renal duct cells

All undergo hyperplasia, esp. when passage system is injured!24 hrs

Renewing Type 3

Urothelia, Mucosal epithelia

All undergo hyperplasia, all times!24 hours

I) All CLC figures are not absolute, depending on the species; 2) Atrophy with age the rat, the liver has the third largest very slow plasma flow rate (12). This the residence time of the xenobiotic concentration is high, with the result 484

Exp Toxic Pathol 44 (1992) 8

tissue volume and a means that in the rat is longer, while its that exposure condi-

tions are maximized in the rat. The possibility of biotransformation and detoxification overload is increased. The liver is also unique among organs in rat and man in that the liver receives xenobiotics mainly via the portal circulation at

Table 5. Comparative Data on Tissue Volume and Plasma Flow Rate from a 500 g Rat l) and a 70,000 g Human 2).

Rat: Weight 500 g, Surface 812 cm2

Skeletal Muscle Adipose Tissue Liver Plasma Intestine Kidneys

Man: Weight 70,000 g, Surface 8,000 cm 2

Tissue Volume (ml)

Rank

Plasma Flow Rate (rul/min)

Rank

Tissue Volume (ml)

Rank

Plasma Flow Rate (rul/min)

Rank

245.0 35.0 20.0 19.6 11.2 3.6

1 2 3 4 5 6

22.4 3.6 4.7 85.0 14.6 12.8

2 6 5 1 3 4

25,000 10,000 1,350 3,000 2,100 280

1 2 5 3 4 6

420 200 800 3,670 700 700

4 5 2 1 3 3

Bold faced is correlated ranking; I) Modified from JAIN et al., 1982; 2) Modified from BISHOFF, 1975 72 %, and not exclusively via the systemic circulation at 22 % (4, 11). Diethylnitrosamine (DEN) is a genotoxic, activationdependent agent (table 6). Phenobarbital (PB) increases the intensity of metabolic reactions in the hepatocytes, while creating an abundance of reactive oxygen radical species (table 4). The interplay between DEN and PB as used in the current protocol, maximizes the insult. In the kidney, DEN neoplasia takes a different time-course, because in this organ the constituent cell types are different (table 4). It takes longer for DEN to unfix the genome of these cells because PB does not help in this process. Only nitrilotriacetic acid (NTA) helps by altering the cellular communication channels and the formation of

hydroxy radicals (I, 6). This is accomplished via its chelating effects. Dimethylbenz(a)anthracene (DMBA) is also genotoxic and activation-dependent (table 6). Diethylstilbestrol (DES) is a nonsteroidal compound with a strong estrogenic potency (10). As such it helps DMBA unfix the genome (and level of differentiation) of endocrinocytes (table 4). Through paracrine stimulation DES also enhances the division in endocrinocytes by increasing the production of growth factors and/or decreasing the production of inhibitory cytokines (I). Methylnitrosourea (MNU) is a genotoxic but activationindependent substance (table 6). Its carcinogenic organotropism is centered mainly in the mucosal epithelial of the

Table 6. Identification of Chemical Agents.

Chemical

Diethylnitrosamine (DEN)

Single Dose (mg/kg)

Factor l )

Route

Mode of Action

Target Site

20.4

10

i.p.

Genotoxic agent; Activationdependent

Liver, Kidney

Dimethylbenz(a) anthracene (DMBA)

2.5

14

gavage

Genotoxic agent; Activationdependent

Mammary gland

Methylnitrosourea (MNU)

1.0

30

gavage

Genotoxic agent; Activationindependent

Stomach (nonglandular)

Phenobarbital sodium (PB)

25.0

168

diet

Biotransformation agent

Liver

Nitrilotriacetic acid trisodium monohydrate (NT A) Diethylstilbestrol (DES)

400.0

168

diet

Che1ating agent

Kidney

0.2

24

s.c.

Mammary gland

Butylated hydroxyanisole (BHA)

600.0

168

diet

Bormonal agent with nonsteroidal estradiol activity Antioxidant agent

Stomach (nonglandular)

I) Number of times needed to obtain the cumulative dose Exp Toxic Pathol 44 (1992) 8

485

nonglandular rodent stomach, site of renewing type 3 cells (table 4). The task of unfixing the differentiation of these cells via MNV does not use much energy. Butylated hydroxyanisole (BHA) is an antioxidant agent with prooxidant properties (5, 23). BHA also induces the cytochrome PA50 system (23). Extensive data have been collected on the dose-response profile of each and every one of these positive control test substances. Possession of these data is a very important basic requirement for conducting the ARA. The amount of the compound under investigation (CpdX) when given from weeks 0-14 should come in 3 doses (13). All should be derived from previous studies. The rate of bioavailability at the level of the biophase expressed in the area under the blood level curve (AVC) , the actual peak height and the time to reach the peak of the AVC, as well as the AVC of the target tissue(s) should be determined (11). The expressions of toxicity at the target sites and the range of the dose-reponse should also be available. When the CpdX is given between 14-38 weeks, only the high dose should be used. The duration of the entire assay is 38 weeks. There is an interim sacrifice at 14 weeks, and a second at 26 weeks (table 3). This timeframe represents 39 % of the lifespan of the rat, or 322.5 months (27 years) of human time-equivalents (11). In humans as well as in the rat, this is generally accepted to be the latency period needed for carcinogenesis (9, 10, 17). Prior to sacrifice, bromodeoxyuridine (BrdV) is given for the purpose of monitoring proliferation at the target site. At anytime during the course of the study, semi-quantitative histomorphometry can also be performed. All the standard duringlife, gross and microscopic parameters are recorded and evaluated. This assay is conducted according to the good laboratory practices for nonclinical laboratories of any regulatory agency in Europe, Japan or the V.S.

Discussion and conclusions This accelerated assay has many fundamental advantages. First, it offers a testable hypothesis for each step of the neoplastic process as induced by chemicals. It has the ability to control and modulate each step of this process (23). It has the ability to monitor morphologically each step of the process. It also has the ability to normalize the factors of species-specific genetics, such as age, body size, histokinetics and xenodynamics (11). In this way it yields results that one can extrapolate across species. Because of its duration of 38 weeks and the timing in the overall product development scheme, it serves as a bridge between subchronic toxicity and carcinogenicity. In this way, if the results are positive, the sponsor is spared having to conduct 2 year carcinogenicity studies in rats and mice. Valuable years are likewise saved in product development. Furthermore, the ARA normalizes the factor of age by examining animals before the crop of "spontaneous" neoplasms obscures the "induced" ones, without compromising the unavoidable factors of chronicity and latency in carcinogenesis (11). Likewise, by 486

Exp Toxic Pathol 44 (1992) 8

avoiding body weight gain changes over two years, it normalizes the neoplastic consequences of these changes, since increased body weight increases the tumor burden (23). It also offers appropriate testing conditions by controlling the dose and route of administration, so that dose directly reflects site exposure (13). Consequently, the results are relevant because this assay excludes false negative and false positive results, while it gives appropriate weight to both benign and malignant neoplasms. This increases both the sensitivity and selectivity of this assay (14). Finally, this assay correlates ideally with other shorterterm genetic and paragenetic assays, as well as all other shorter chemical safety assessment and pharmacokinetic studies (11, 20, 23). It is a complementary assay with conclusive results. The power of its predictability is very strong. It combines chronicity with insight into the mechanisms of neoplasia, without taking 3 years to complete. We know of no disadvantages.

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