It has been demonstrated that in humans certain factors such as early menarche, late pregnancy, and nulliparity are associated with a higher risk of developing breast cancer, while early pregnancy acts as a protective factor. Induction of mammary cancer in rats by administration of the chemical carcinogen 7, 12-dimethylbenz(a)anthracene reveals that the same factors influencing human breast cancer risk also affect the susceptibility of the rat mammary gland to the chemical carcinogen. Nulliparous rats and rats undergoing pregnancy interruption are more susceptible to developing carcinomas. This fact has been attributed to the incomplete differentiation of the gland at the time of carcinogen administration. Parous rats are resistant to the carcinogenic effect of DMBA, which is explained by the complete development of the gland attained during pregnancy and lactation. This development is manifested by the differentiation of terminal end buds into secretory units, which have a smaller proliferative compartment; the epithelial cells of these secretory units have a longer cell cycle, less avidity for binding DMBA, and possess a more efficient DNA excision repair capacity.

Differentiation of the mammary gland and susceptibility to carcinogenesis

The covalent binding of chemical carcinogens to DNA of mammalian organs is expressed per unit dose, and a 'Covalent-Binding Index', CBI, is defined. CBI for various carcinogens span over 6 orders of magnitude. A similar range is observed for the carcinogenic potency in long-term bioassays on carcinogenicity. For the assessment of a risk from exposure to a carcinogen, the total DNA dmaage can be estimated if the actual dose is also accounted for. A detailed description is given for planning and performing a DNA-binding assay. A complete literature survey on DNA binding in vivo (83 compounds) is given with a calculation of CBI, where possible, 153 compounds are listed where a covalent binding to any biological macromolecule has been shown in vivo or in vitro. Recent, so far unpublished findings with aflatoxin M1, macromolecule-bound aflatoxin B1, diethylstilbestrol, and 1,2-epithiobutyronitrile are included. A comparison of CBI for rat-liver DNA with hepatocarcinogenic potency reveals a surprisingly good quantitative correlation. Refinements for a DNA-binding assay are proposed. Possibilites and limitations in the use of DNA binding in chemical carcinogenesis are discussed extensively.

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In vivo covalent binding of organic chemicals to DNA as a quantitative indicator in the process of chemical carcinogenesis.

The susceptibility of the rat mammary gland to carcinogenesis decreases with aging and is nullified by pregnancy and lactation. This work was carried out to determine whether these differences in susceptibility to carcinogenesis are the result of variations in cell kinetics induced by both aging and parity. Cell cycle (Tc) and growth fraction (GF) were studied in the mammary gland epithelium of young virgin, old virgin, and parous Sprague-Dawley rats. For the study of Tc, five young virgin, five old virgin, and five parous rats received while in estrus an i.p. injection of 1.7 muCi [3H] thymidine per g body weight. Mammary gland biopsies were taken at 2-hr intervals for the first 24 hr and every 4 hr thereafter up to 72 hr. GF was determined in two young virgin, two old virgin, and two parous rats in which an osmotic minipump releasing 1 microCi [3H]thymidine per hr was implanted i.p. After 5 days, the animals were killed, and the mammary glands were removed. Mammary gland biopsies and whole glands were fixed in Bouin's fluid and embedded in paraffin. Deparaffinized sections were processed for autoradiography. The labeled mitosis wave, labeling index, and GF were determined. The results indicated that, in young virgin rats, mammary gland Tc was 9.9 hr in the most undifferentiated structures (terminal end buds), lengthening to 17.3 hr in terminal ducts (TD) and to 28.2 hr in alveolar buds (AB). In the mammary gland of old virgin rats, Tc was 18.75 hr in terminal ducts in regression, 20.57 hr in TD, and 30.75 hr in AB. In parous rats, mammary gland Tc was 23.9 hr in TD and 49.63 hr in AB. Lengthening of Tc in all of these structures was due to a lengthening of G1, while the duration of the other phases of the cell cycle remained unchanged. GF was 0.55 in terminal end buds of young virgin rats, decreasing to 0.39 and 0.13 in TD and AB, respectively. In terminal ducts in regression of old virgin rats, GF was 0.19, decreasing to 0.054 and 0.030 in TD and AB, respectively. In TD and AB of parous rat, mammary gland GF was 0.009 and 0.004, respectively. These results suggest that the high susceptibility to carcinogenesis that has been demonstrated in young virgin rats is due to the presence of a large proliferative compartment, mainly in terminal end buds and TD, while the low susceptibility of parous animals is due to the formation of a large compartment of nonproliferating cells, and that those cells still proliferating have a longer G1 than do those of young and old virgin rats.

https://cancerres.aacrjournals.org/content/canres/40/8_Part_1/2677.full.pdf

Influence of Differentiation and Cell Kinetics on the Susceptibility of the Rat Mammary Gland to Carcinogenesis

Carcinogenesis by aromatic amines

Efforts in estimating carcinogenic risk in humans from long-term exposure to chemical carcinogens have centered on the problem of low-dose extrapolation. For chemicals with metabolites that interact with DNA, it may be more meaningful to relate tumor response to the concentration of the DNA adducts in the target organ rather than to the applied dose. Many data suggest that the relation between tumor response and concentration of DNA adducts in the target organ may be linear. This implies that the nonlinearities of the dose-response curve for tumor induction may be due to the kinetic processes involved in the formation of carcinogen metabolite--DNA adducts. Of particular importance is the possibility that the kinetic processes may show a nonlinear "hockey-stick" like behavior which results from saturation of detoxification or DNA repair processes. The mathematical models typically used for low-dose extrapolation are shown potentially to overestimate risk by several orders of magnitude when nonlinear kinetics are present.

https://science.sciencemag.org/content/sci/219/4588/1032.full.pdf

Implication of nonlinear kinetics on risk estimation in carcinogenesis

Summary The O -sulfonate of N -hydroxy-2-acetylaminofluorene was not formed in vitro in the mammary gland or in the sebaceous gland of the external auditory canal (Zymbal9s gland), tissues of the rat that are highly susceptible to carcinogenesis by 2-acetylaminofluorene and N -hydroxy-2-acetylaminofluorene. Consequently, the O -sulfonate of N -hydroxy-2-acetylaminofluorene, reported to be an ultimate reactive metabolite of these carcinogens in male rat liver, does not appear to be involved in the mechanism of carcinogenesis in all tissues of the rat in which tumors are induced by these compounds.

https://cancerres.aacrjournals.org/content/canres/31/4/387.full.pdf

Lack of N-Hydroxy-2-acetylaminofluorene Sulfotransferase Activity in the Mammary Gland and Zymbal's Gland of the Rat

Sprague-Dawley female rats were sacrificed at various times after the intragastric administration of 7,12-dimethylbenz(a)anthracene (DMBA)-3H. The abdominal-inguinal mammary glands were removed, and the parenchymal cells were separated. Parenchymal cell DNA and protein were isolated, and the DNA was purified by density equilibrium centrifugation in cesium chloride. Sixteen hr after carcinogen feeding, DMBA binding amounted to 47 pmoles/mg of DNA. Fifty % of the DMBA bound to DNA at 16 hr was present 14 days after administration, and 31% was still detectable at 42 days. The amount of DMBA binding to protein was less than one-half that observed bound to DNA. Furthermore, by 14 days, DMBA binding to protein had declined to just detectable levels. Increased parenchymal cell DNA content, due to cellular proliferation resulting from DMBA-induced tumorigenesis, was not responsible for the decreased DNA specific activity observed at 14 and 42 days. Thus, persistent binding of DMBA to rat mammary parenchymal cell DNA in vivo was demonstrated.

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The binding of 7,12-dimethylbenz(a)anthracene to mammary parenchyma DNA and protein in vivo.

The binding of 7,12-dimethylbenz[a]anthracene (DMBA) to mammary gland and liver DNA of female Sprague-Dawley rats either 35, 50, or 120 days of age at the time of carcinogen administration was studied. Following a single oral feeding of tritium-labeled DMBA, the level of binding to liver DNA of rats in all 3 age groups was significantly lower, at all times during a 6-week period, than that of binding to mammary DNA. The amount of DMBA bound to liver DNA was a function of the amount of carcinogen administered and not the age of the animal. In contrast, DMBA binding to mammary DNA was dependent on the age of the animal at the time of carcinogen feeding. Furthermore, in the age group with 100% tumor induction (50 days old), DMBA binding increased directly with the amount of carcinogen fed; this was not the case for the other 2 age groups. These results indicated that a significant correlation existed between the age of the rat, the amount of DMBA bound to DNA, and the incidence of mammary tumors following carcinogen feeding.

Douglas H. Janss

Papers

Lack of N-Hydroxy-2-acetylaminofluorene Sulfotransferase Activity in the Mammary Gland and Zymbal's Gland of the Rat

The binding of 7,12-dimethylbenz(a)anthracene to mammary parenchyma DNA and protein in vivo.

Age-Related Modification of 7,12-Dimethylbenz[a]anthracene Binding to Rat Mammary Gland DNA

Aryl Hydrocarbon Hydroxylase and Epoxide Hydratase Activities: Age Effects in Mammary Epithelial Cells of Sprague-Dawley Rats

Radioactivity in rat mammary gland after the administration of 2-acetylaminofluorene-9- 14 C and its N-hydroxy metabolite.