Rodent 2-year cancer bioassays and in vitro and in vivo genotoxicity tests insufficiently predict risk or model development of human carcinomas

Abstract

In 2018, the National Institutes of Health estimated that 1,735,350 new cancer cases were diagnosed in the United States and 609,640 patients died from various forms of cancer. The vast majority of those cancer deaths occurred from cancers derived from epithelial cells, that is, carcinomas. The most lethal cancers in American women based upon estimated deaths occurred in the lung and bronchus (352,209), breast (205,675), colon and rectum (123,617), pancreas (96,571), and ovary (71,141). The largest cancer killers in men occurred in the lung and bronchus (427,587), prostate (140,086), colon and rectum (135,542), pancreas (100,599), and liver (80,526). Histologically different carcinomas are initiated, clonally expanded, and progressed in association with particular changes in genes and also epigenetic alterations. Currently employed genotoxicity testing protocols emphasize testing for the initiating (mutational) potential of the test agent. While 2-year chronic rodent cancer bioassays test for the entire spectrum of carcinogenic transformation and development, the high doses used in these assays induce cytotoxicity leading to increased cellular proliferation rates and high false-positive rates of tumor induction in non-genotoxic chemicals. The low cancer induction from high radiation exposures experienced by atomic bomb survivors in Hiroshima and Nagasaki, Japan, and the epidemiological evidence showing that cigarette smoking duration and not intensity is associated with lung cancer risk both support a more important role for tumor promotion rather than initiation in the clinical presentation of human carcinomas. Cancer hazard assessment testing protocols and weight-of-the-evidence analysis of agent-specific cancer risk should be better aligned with the pathogenesis of human carcinoma.

Keywords

Genotoxicity protocols rodent cancer bioassays clinical relevance cancer assessment pathogenesis of human carcinoma

Executive summary

A number of concepts will be developed and supported including the following:

The major cancer killers in the United States in both women and men are carcinomas, that is, cancers of epithelial origin.

Malignant carcinomas develop when a single epithelial cell undergoes genetic change allowing it to escape normal growth regulatory pathways. Additional genetic changes are required to clonally expand this initially transformed epithelial cell, convert the slowly proliferating primary tumor from a relatively poorly vascularized to a more vascularized state, and ultimately to an invasive or metastatic carcinoma. Both mutations and epigenetic changes can play an important role in carcinogenesis.

Clinical observations of the carcinogenicity of workplace chemicals predated conduction of the first animal cancer bioassays.

The life cycle of malignant tumor development can be modeled in the two-stage mouse dermal promotion assay as going through the stages of tumor initiation, promotion, and progression. Usage of the terms initiation, promotion, and progression derive from observations in this assay.

In humans, carcinogenesis also advances in molecular stages analogous to tumor initiation, promotion, and progression. While the molecular changes cannot be visualized, sequential histological stages of carcinoma development including hyperplasia (increase in cell number), dysplasia (precancerous stage), carcinoma in situ (not yet invasive or noninvasive carcinoma), invasive carcinoma, and metastatic carcinoma result from these molecular changes.

Current genotoxicity testing protocols including the Ames Salmonella bacterial mutagenicity assay, CA assay, micronucleus (MN) assay, Comet assay, Big Blue Transgenic Rodent (TGR) Mutation Assay, and hypoxanthine-guanine phosphoribosyl transferase (HGPRT assay) test for DNA damage analogous to tumor initiation rather than promotion (clonal expansion) or progression (carcinoma in situ to invasive carcinoma or metastatic carcinoma).

Although no in vitro test can model the entire carcinogenic pathway, an assay that appears to model the early stages of human cancer development is the human–hamster hybrid A_L assay with embryonic chick cell cocultivation for metabolic activation developed at the Eleanor Roosevelt Cancer Research Institute, Colorado State University, and Dartmouth Medical School.

Despite containing a large number of initiating agents (genotoxic mutagens and IARC (International Agency for Research on Cancer) carcinogens), extensive epidemiological evidence demonstrates that lung cancer risk from smoking is mainly related to tumor promotion.

Estrogen can function as a tumor promoter at several tissue sites in women.

Prostate cancer requires promotion to present as clinically significant pathology as small asymptomatic prostate cancers are common in men over 50.

Obesity increases the risk of developing several different cancers. Several different mechanisms including low-level chronic inflammation and alterations in hormones probably underlie this association.

Radiation is a mutagen (initiating agent). The atomic bomb survivors in Hiroshima and Nagasaki show relatively low cancer rates. These relatively low cancer rates suggest that the initiating mutations, when not followed by promotional clonal expansion, do not sometimes result in development of cancer. The bomb survivor data are consistent with experimental results reported by Kiraly et al.¹ where mutations alone led to low levels of tumor development in rodents. In contrast, the same mutations in the presence of cellular proliferation displayed a high level of tumor development.

National Toxicology Program (NTP) studies do not appropriately evaluate genotoxicity and exhibit a high false-positive rate.

Cancer hazard assessment of an industrial chemical using an evaluation of possession or absence of structural elements of carcinogenicity, and noninvasive exposure studies of relevant occupational cohorts, is more data-based, and more cost-effective, than reliance on the current battery of in vitro genotoxicity assays and the 2-year rodent cancer bioassay. In addition, in situations where an in vitro genotoxicity evaluation might be advantageous, mutagenicity assessed in the human–hamster hybrid A_L assay with embryonic chick cell cocultivation for metabolic activation is mechanistically consistent with current hypotheses regarding tumor initiation.

Biggest cancer killers in the United States are carcinomas

In 2018, an estimated 1,735,350 new cases of cancer were diagnosed in the United States and 609,640 people died from cancers occurring at various organ sites and histological presentations.^2,3 Primarily due to higher historical rates of smoking and drinking excess alcohol, cancer mortality is higher among men than women (196.8 per 100,000 men and 139.6 per 100,000 women). Cancer incidence in the United States also differs significantly by ethnicity with cancer mortality highest in African American men (239.9 per 100,000) and lowest in Asian/Pacific Islander women (88.3 per 100,000).^2,3 Table 1 shows that the most lethal cancers in American women based upon estimated deaths occurred in the lung and bronchus (352,209), breast (205,675), colon and rectum (123,617), pancreas (96,571), and ovary (71,141). The largest cancer killers in men occurred in the lung and bronchus (427,587), prostate (140,086), colon and rectum (135,542), pancreas (100,599), and liver (80,526). Table 2 illustrates that the vast majority of both cancer cases and cancer deaths in the United States are due to carcinomas of epithelial origin, rather than sarcomas deriving from bone, connective, or muscle tissue.⁴

Table 1.

Cancer deaths by primary cancer site in the United States (2011–2015).^a

All races
Site	Both sexes	Males	Females
Oral cavity and pharynx	45,589	32,263	13,326
Esophagus	73,930	59,082	14,848
Stomach	56,128	33,487	22,641
Colon and rectum	259,159	135,542	123,617
Liver and intrahepatic bile duct	119,070	80,526	38,544
Pancreas	197,170	100,599	96,571
Larynx	18,643	14,897	3746
Lung and bronchus	779,796	427,587	352,209
Melanoma of the skin	45,982	30,225	15,757
Breast	207,915	2240	205,675
Cervix uteri	20,673	0	20,673
Corpus and uterus, NOS	46,700	0	46,700
Ovary	71,141	0	71,141
Prostate	140,086	140,086	0
Testis	1934	1934	0
Urinary bladder	78,045	55,652	22,393
Kidney and renal pelvis	69,348	45,076	24,272
Brain and other nervous system	77,375	43,423	33,952
Thyroid	9013	4081	4932
Hodgkin lymphoma	5585	3247	2338
Non-Hodgkin lymphoma	101,359	56,402	44,957
Myeloma	59,376	32,222	27,154
Leukemia	116,463	67,201	49,262
Others	331,289	174,767	156,522
All sites	2,931,769	1,540,539	1,391,230

NOS: not otherwise specified.

^aAdapted from USDHHS.²

Table 2.

Ratio of carcinomas/sarcomas for cancers of different sites.

Site	% carcinoma/% sarcoma
Oral cavity and pharynx	90/10
Esophagus	90/10
Stomach	95/5
Colon and rectum	95/5
Liver and intrahepatic bile duct	98/2
Pancreas	95/5
Larynx	95/5
Lung and bronchus	98/2
Melanoma of the skin	Neither (basal cell)
Breast	98/2
Cervix uteri	95/5
Corpus and uterus	90/10
Ovary	90/10
Prostate	98/2
Testis	95/5
Urinary bladder	96/4
Kidney and renal pelvis	96/4
Brain and other nervous system	Not always applicable
Thyroid	98/2
Hodgkin lymphoma	Neither
Non-Hodgkin lymphoma	Neither
Myeloma	Neither
Leukemia	Neither
Others	Undefinable

Genetic and epigenetic basis of cancer development

Many malignant tumors start out as a single normal cell that undergoes a specific mutation(s) that transforms that cell to a cancer cell, that is, a variety of malignant tumors are monoclonal in origin⁵ although some lymphoid tumors are composed of different cell types. Tumors achieving a size sufficient for detection by a physician contain several billion cells.⁶ The transformational event occurs when a cell acquires specific growth advantages through the stepwise accumulation of heritable changes in gene function.⁷ Normal cells are only capable of dividing a specific number of times in cell culture before entering into apoptosis and undergoing programmed cell death.⁸ In contrast, transformed cells can divide an indefinite number of times in cell culture.⁸

The process of transforming a normal cell into a malignant tumor is frequently directed by alterations in two different classes of genes: (1) tumor suppressor genes that inhibit cell growth and survival and (2) oncogenes that promote cell growth and survival. Several consecutive genetic alterations are usually required for a malignant tumor to fully develop.⁹

The malignant phenotype is determined by the interaction of tumor suppressor genes and oncogenes.⁷ Alteration in the protein-encoding nucleotide template can amplify the number of copies of a particular cancer gene, or increase the transcription by that gene.⁷ Table 3 shows some of the mutations associated with the major human carcinomas.¹⁰

Table 3.

Mutations seen in major human carcinomas.^a

Organ	Inherited gene mutations associated with tumors
Breast	BRCA1 or BRCA2 gene; TP53 gene
Ovary	BRCA1 or BRCA2 gene; MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2.
Endometrium	MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2.
Fallopian tube	BRCA1 or BRCA2 gene
Primary peritoneal	BRCA1 or BRCA2 gene
Pancreas	BRCA2 gene; MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2.
Prostate	BRCA2 gene
Stomach	MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2.
Small intestine	MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2.
Ureter	MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2.
Brain	MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2; TP53 gene
Kidney	MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2.
Bile duct	MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2.
Colon	MMR genes MLH1, MSH2, MSH6, PMS1, and PMS2.
Lung	TP53, EGFR, and K-ras

MMR: mismatch repair; EGFR: epidermal growth factor receptor.

^aAdapted from ACS.¹⁰

Epigenetic changes are heritable changes in gene expression activity that occur without alteration in DNA sequences but are sufficiently robust as to regulate gene expression.¹¹ Both genetic and epigenetic modifications of DNA contribute to cancer development.^12

–17 Tumor initiation and progression appear to be predominantly driven by sequential mutations. However, microenvironment-mediated epigenetic perturbations play important roles in neoplastic development.¹⁸ Key processes responsible for epigenetic regulation include DNA methylation, modifications in chromatin (covalent modification of core histones), nucleosome positioning (physical alteration), and posttranscriptional gene regulation by noncoding RNA (microRNAs).¹⁵ A number of well-characterized epigenetic modifications have been linked to aberrant gene functions and altered gene expression. Table 4 shows some of the epigenetic modifications that play critical roles in the pathobiology of cancer.

Table 4.

Epigenetic changes causally associated with human cancer.

Cancer type	Epigenetic changes	Example	Reference
Prostate	Epigenetic markers changes; DNA methylation patterns; histone marks	GSTP1 gene in prostate cancer	Esteller¹⁹
Colon	Comparative patterns; changes in epigenetic markers over time	p16INK4a gene in colon cancer	Esteller²⁰
Brain or spine	Methylation and gene expression profiles	MGMT gene in glioma	Esteller²¹
Esophageal	Hypermethylation-induced silencing of transcription factors	RUNX3 gene	Sharma et al.²⁰; Long et al.²¹
Colorectal and gastric	Hypermethylation-induced silencing of transcription factors	GATA-4 and GATA-5 genes	Sharma et al.²⁰; Akiyama et al.²²
Breast	Silencing of DNA repair genes	MLH1, BRCA1 genes	Sharma et al.²⁰

GSTP1: glutathione S-transferase P; MGMT: O6-methylguanine-DNA methyltransferase.

In 1990, Fearon and Vogelstein⁹ provided an elegant demonstration of how sequential mutations and epigenetic changes interact to form a human cancer, that is, colorectal cancer (CRC). Figure 1 summarizes the CRC pathway as elucidated by Fearon and Vogelstein.⁹ In the CRC pathway, tumorigenesis proceeds through a series of genetic alterations involving oncogenes (ras) and tumor suppressor genes (particularly those on chromosomes 5q, 17p, and 16q). Three stages of adenomas represent tumors of increasing size, dysplasia, and villous content.

Figure 1.

Summarization of the CRC pathway.⁹ CRC: colorectal cancer.

A mutation on chromosome 5q is inherited in patients with familial adenomatous polyposis. This mutation might cause the hyperproliferative epithelium in these patients. In patients without polyposis, tumors are associated with a loss or mutation of the same section of DNA on chromosome 5q occurring at a relatively early stage of tumorigenesis. In patients with or without polyposis, hypomethylation is seen in very small adenomas. This hypomethylation may lead to aneuploidy thereby resulting in the loss of suppressor gene alleles.

A single cell of a preexisting small adenoma can incur a ras gene mutation (usually K-ras) that facilitates clonal expansion and produces a larger and more dysplastic tumor. The chromosomes most frequently deleted include 5q, 17p, and 18q. The tumor suppressor gene is the putative target of the loss event. The relative timing of the chromosome loss event may be important.

Allelic deletions of chromosomes 17p and 18q usually occur at a later stage of tumorigenesis than do deletions of chromosome 5q or ras gene mutations. The sequential order of these changes is variable. The net effect of accumulation of these changes, rather than their sequential order, seems to be most important. After carcinomas have formed, the tumor progression process continues. Accumulated loss of suppressor genes on additional chromosomes correlates with metastatic potential and expiration of the patient.²³

Historical role of cancer bioassays

The original investigations of workplace-related cancers were initiated by the observations of unusual tumors in association with certain occupations²⁴ including chimney sweeps,^25,26 mule spinners,^25,27,28 aniline dye workers,²⁹ clock workers using radium-impregnated dye,³⁰ and chemical workers exposed to benzene.³¹ Following detection in humans, the causative agents were later studied in animals.^32

–37

In general, the ongoing efforts to remove carcinogens from the workplace in developed countries have resulted in amelioration of exposure.³⁸ The paradigm of testing for chemical induction of cancer in rodents has shifted from confirmation of observation to intended prophylaxis. The misperceptions resulting from ahistorical presentism have inverted the chronological sequence of clinical observation and animal testing and inappropriately elevated animal testing to a preeminent role in detecting rather than describing environmental carcinogens.³⁹ Table 5 shows a representative subset of IARC-classified agents that were recognized as cancerous (or potentially cancerous) prior to testing in any animal.^{40

–68} The average time lapsing from first reported observation or collection of formal epidemiological data to conduction of the first animal tests is 64 years (range 9–387 years). The intent of this animal testing was to provide data to substantiate that the suspect agents were indeed dangerous and that certain manufacturing, handling, shipping, and storage precautions and/or regulations were needed for the safety of workers and consumers.

Table 5.

IARC agents where clinical observation or epidemiological cancer data preceded animal cancer testing.

IARC agents	IARC Group	Earliest year for epidemiological study of cancer data (latency periods 10–30 years)	Earliest year tested for cancer with rodents/rabbits	References
Asbestos	1	1907	1962	Albin et al.⁴⁰; Wagner⁴¹
Beryllium	1	1937	1946	Mancuso and El-Attar⁴⁶; Mancuso^43 –45; Gardner and Heslington⁴²
Cadmium	1	1923	1961	Potts⁴⁷; Haddow et al.⁴⁸
1,3-Butadiene	1	1964	1984	McMichael et al.⁴⁹; NTP⁵⁰
Bis(chloromethyl) ether	1	1956	1975	Thiess et al.⁵¹; Kuschner et al.⁵²
Ethylene oxide	1	1925	1981	Steenland et al.⁵³; Dunkelberg⁵⁴; Swaen et al.⁵⁵
Glass wool	3	1933	1981	Boffetta et al.⁵⁶; Lee et al.⁵⁷
Sulfur mustard	1	1914	1950	Case and Lea⁵⁸; Heston⁵⁹
Radon gas	1	1556	1943	Arnstein⁶⁰; Rajewsky et al.⁶¹; Rostoski et al.⁶²
Crystalline silica	1	1942	1983	Rice et al.⁶³; Holland et al.⁶⁴
Vinyl chloride	1	1939	1978	Smulevich et al.⁶⁵; Lee et al.⁶⁶
TCDD	1	1953	1977	Van Miller et al.⁶⁷; Ott and Zober⁶⁸

IARC: International Agency for Research on Cancer.

In addition to transposing the chronological sequence of clinical observation and animal studies, the degree of concordance between rodents and humans regarding pulmonary carcinoma has been overstated.³⁹ In 2-year NTP inhalation studies, Smith and Anderson⁶⁹ have shown that concordance of pulmonary tumor formation between phylogenetically similar rats and mice is low, thereby questioning the potential degree of concordance between much more phylogenetically distant rodents and humans. Krewski⁷⁰ has reported the concordance between humans and mice for lung tumors associated with IARC Group 1 (known human) carcinogen exposure. Using a κ statistic at 90% confidence, IARC Group 1 agents showed higher concordance than did Group 2a, 2b, and Group 3 chemicals and mixtures. The κ value for the concordance between humans and mice in Group 1 chemicals was 0.17 (−0.2 to 0.53). A κ value of between 0.01 and 0.20 represents only a slight concordance. The concordance (human vs. mouse) for lung tumors was slight and equal to tumors of the urinary system (0.12 (−0.05 to 0.12)). The concordance (human vs. mouse) for all other organ systems was higher than the lung system (κ 0.51–0.64 = moderate to substantial).

Two-stage mouse dermal promotion assay—initiation, promotion, progression

There are several separate but interrelated conceptual frameworks for ideating the life cycle of cancer from its origins as a single normal cell to its final stage as a large mass of billions of locally invasive or metastatic cells within a malignant tumor. One of the most useful conceptual frameworks has been the two-stage mouse dermal promotion assay from which the terms initiation, promotion, and progression derive.^71,72 The eponymous term “initiation” refers to the first step in the mouse dermal promotion assay.⁷³ A parent chemical that reacts with DNA can serve as an initiator, although metabolic enzymes are frequently required to convert a nonreactive parent chemical into an electrophilic mutagen.⁷⁴ Different tissues and different species can possess significantly different levels of metabolic enzyme activity. This differential in metabolic potential confers tissue and species specificity on many initiators.⁷⁵

It is important to understand that the effects of initiators are reversible as long as repair occurs prior to a cell division. After the DNA in a particular cell has been altered by a chemical initiating agent, that cell is susceptible to promotion until its death. Daughter cells produced from the division of the mutated cell will also carry the mutation.⁷⁴ A linear relationship has been observed between the dose of initiator and the quantity of tumors produced in studies of mouse skin carcinogenesis. Therefore, any exposure to an initiator increases cancer risk and this risk increases with higher levels of exposure.⁷⁵

Promotion refers to the process of enhancing cellular proliferation.⁷⁶ Cells mutated by an initiator are susceptible to the effects of promoters. Promotion gives rise to a large number of daughter cells containing the original mutation induced by the initiating chemical.⁷⁷ Promoters do not affect cells not previously treated with an initiator.⁷⁵ Unlike initiators, promoters do not covalently bind to DNA or cellular macromolecules. Promoters can be categorized as specific or nonspecific. Specific promoters interact with receptors on or in target cells of defined tissues. Nonspecific promoters alter gene expression without binding to a known receptor.⁷⁴ Tissue and species specificity results from the differential distribution of receptors on various tissue types. The effect on tumor growth of promoter application is dose-dependent. However, promotion displays both threshold and ceiling effects with both a minimum dose required and a maximum dose beyond which no further effects are seen.⁷⁵

Progression refers to the stepwise transformation of a benign tumor to a malignant tumor. Repeated application of a promoting agent onto previously initiator-treated mouse skin can produce benign papillomas. After cessation of treatment with promoter, most of these papillomas regress but some progress to cancer.⁷⁸ Benign papillomas that progress to cancer have acquired an additional, spontaneous mutation.⁷⁹ Progression is associated with karyotypic change as virtually all benign tumors that advance to malignant tumors become aneuploid (incorrect number of chromosomes). Karyotypic change is associated with increased growth rate, invasiveness, metastatic potential, and biochemical and morphological changes.⁷⁵

In humans, molecular stages analogous to tumor initiation, promotion, and progression are concomitant with the sequential histological stages of carcinoma development

All of the major human carcinomas pass through histological stages of hyperplasia, metaplasia, dysplasia, carcinoma in situ, and invasive carcinoma.⁸⁰ Cancers of the central nervous system, bone, blood, and lymph system do not use the same terminology due to differences in pathology. In humans, molecular stages analogous to tumor initiation, promotion, and progression are concomitant with but variably overlapping with the sequential histological stages of carcinoma development including hyperplasia (increase in cell number), dysplasia (precancerous stage), carcinoma in situ (not yet invasive or noninvasive carcinoma), invasive carcinoma, and metastatic carcinoma. Figures 2 to 6 demonstrate the various stages of cancer development for breast and colon cancer, that is, normal tissue, carcinoma in situ, and invasive carcinoma. Figure 2 shows a normal breast lobule and ducts at 4× objective (40×). Figure 3 is an image of invasive ductal carcinoma on the left and ductal carcinoma in situ on the right of the breast at 4× objective (40×). Figure 4 shows normal colonic mucosa at 10× objective (100×). Figure 5 is a photo of a tubular adenoma with adenomatous change in glands at the top of the image and normal glands at the bottom magnified at 10× objective (100×). Figure 6 shows invasive colonic adenocarcinoma on the left and normal colonic mucosa on the right at 40× objective (400×).

Figure 2.

Normal breast lobule and ducts—4× objective (40×).

Figure 3.

Invasive ductal carcinoma on the left and ductal carcinoma in situ on the right of the breast—4× objective (40×).

Figure 4.

Normal colonic mucosa—10× objective (100×).

Figure 5.

Tubular adenoma—adenomatous change in glands at the top of the image and normal glands at the bottom—10× objective (100×).

Figure 6.

Invasive colonic adenocarcinoma on the left and normal colonic mucosa on the right—40× objective (400×).

Current genotoxicity testing protocols test for DNA damage analogous to tumor initiation rather than promotion (clonal expansion) or progression (carcinoma in situ advancing to invasive carcinoma or metastatic carcinoma)

Ames Salmonella mutagenicity assay

The Ames Salmonella mutagenicity assay is the most widely used in vitro genotoxicity assay. The Ames test is relatively inexpensive, reproducible, and has an extremely large historical database of chemicals tested in the assay whose results are available online.⁸¹ In addition, the assay can be conducted with and without metabolic activation by rat liver or human S9 microsomal enzymes.⁸² The mutations measured by the Ames test represent relatively small alterations to the DNA including base-pair substitutions detectable in bacterial strains hisG46, TA100, and TA1535; frameshift mutations of different types detectable in bacterial strains hisD3052, TA98, TA1538, hisC3076, TA1537, hisD6610, and TA97 (Table 6).⁸³ These small Ames mutations are analogous to initiation events in the tumor formation pathway.

Table 6.

DNA sequence specificity on the Salmonella tester strains.^a

Allele/strains	DNA target	Reversion event
hisG46	-G-G-G-	Base-pair substitution
TA100
TA1535

hisD3052	-C-G-C-G-C-G-C-G-	Frameshifts
TA98
TA1538

hisC3076	+1 frameshift	Frameshifts
TA1537	(near -C-C-C- run)

hisD6610	-C-C-C-C-C-C-	Frameshifts
TA97	(+1 cytosine at run of C’s)

hisG428	TAA (ochre)	Transitions/transversions
TA102
TA104

^aAdapted from Mortelmans and Zeiger.⁸³

Chromosome aberration assay

Different mechanisms for repairing DNA double-strand breaks generate a wide spectrum of DNA changes including microscopically visible chromosomal aberrations (CA). Active chromatin is susceptible to CA breakpoints. The distribution of interchromosomal and intrachromosomal breakpoints is generally not random. The arrangement of chromosomes in the interphase nucleus, and differential sensitivity of chromosomes to test agents, can affect the formation of CA. Telomeres and interstitial telomere-repeat-like-sequences play a major role in the formation of CA with sub-telomeric regions serving as hot spots for the formation of symmetrical exchanges between homologous chromatids.⁸⁴ While CA represent a larger disruption to DNA than Ames mutations, positive Ames test results paradoxically correlate more strongly than do positive CA results in predicting the development of rodent tumors.⁸⁵ CA are analogous to an initiating event in the carcinogenesis pathway.

MN assay

The MN assay detects cytogenetic damage. MNs develop when complete chromosomes or chromosomal fragments do not incorporate into the daughter nuclei during the anaphase of cell division but instead remain in the cytoplasm throughout the life cycle of the cell.⁸⁶ MN assays are preferred for assessing chromosome damage because both chromosome loss and chromosome breakage can be measured reliably.⁸⁷ Although chromosome damage represents a much larger insult to DNA than do the base-pair substitutions, frameshifts, transitions, and transversions measured in the Ames test, the MN test does not add to the power of the Ames test to predict the development of rodent tumors.^88
–90 MN assays detect a type of DNA damage akin to initiation in the carcinogenesis process.

Comet assay

Single cell gel electrophoresis assays for the detection of DNA damage in individual cells are referred to as the Comet assay, first used to detect double-stranded DNA breaks induced by X-rays.^86,91,92 Current versions of the Comet assay can detect different types of DNA damage induced by small molecule test agents including single- and double-stranded breaks, alkali-labile sites, oxidative base damage, and DNA cross-linking. The name Comet assay comes from the appearance of the cleaved DNA fragments following forcible migration from the nucleoid cores by an electrophoretic field and after staining with a DNA-specific fluorescent dye. The comet tail length is the distance that the DNA has migrated from the core. Tail length positively correlates with amount of DNA damage and can be used to assess double-stranded DNA damage.^86,93 An additional measure of degree of DNA damage is the size and staining intensity of the comet tail.^86,94 Results from the Comet assay are sometimes used as a tiebreaker to assess the genotoxicity of a chemical agent for which there are contradictory Ames test results. Although the historical database of agents tested in the Comet assay is growing rapidly, it is currently much smaller than the historical database of Ames data.⁹¹ Similar to the Ames test, the DNA damage measured by the Comet assay is analogous to initiation and not promotion or progression.

Big Blue TGR Mutation Assay

The Big Blue TGR Mutation Assay is an in vivo gene mutation assay that can be conducted in mice or rats.⁹⁵ Multiple copies of recoverable target genes are integrated into the genome of Big Blue mice via breeding. The process starts by microinjecting the lambda shuttle vector containing the cII gene into the pronucleus of fertilized eggs from either C57BL/6 mice or Fischer 344 rats. The lambda shuttle vector can be recovered from genomic DNA and mutations in the lambda cII gene can be measured.^96,97 Animals are usually dosed by oral gavage for 28 days. For the 3 days following the 28th day, the animals are not dosed to allow fixation of DNA lesions as stable mutations. On day 31 following mutational stabilization, tissues are collected and high molecular weight DNA is extracted from tissues of interest and purified, the shuttle vector recovered, packaged into phage, infected onto Escherichia coli, and plated for plaque formation at two temperatures. All phages form plaques at 37°C while cII mutant phages form plaques only at 24°C. The number of mutant plaques divided by the total number of plaques gives the mutation frequency.^96,97 The results of this assay are also relevant to the initiation phase of tumor formation, and not to the promotional clonal expansion or progression phases of tumor development.

HGPRT mammalian mutation assay

A locus refers to a particular place on a chromosome. A locus in Chinese hamster ovary (CHO), V79, and L5178Y cells has been identified that controls the expression of the enzyme HGPRT. Forward mutations at this locus can be measured by the HGPRT mammalian mutation assay.⁹⁸ The HGPRT assay can be used to detect and measure point mutations involving base substitutions, deletions, frameshifts, and rearrangements within the locus. The assay can be performed with and without metabolic activation by rat liver microsomal enzyme fraction S9.⁹⁹ The type of DNA damage measured by the HGPRT assay is analogous to tumor initiation and not tumor promotion or progression.

Uniqueness of mutagenicity evaluation by the human–hamster hybrid A_L assay with embryonic chick cell cocultivation for metabolic activation

Our research group has extensively examined the entire NTP database up through 2018.^{69,85,100

–104} The correlation between genotoxicity and clastogenicity results and development of tumors in either rats or mice can be calculated from the data in the NTP database. In contrast, the degree of correlation between the genotoxicity test results or rodent tumor data and human cancer risk cannot be determined. Therefore the need exists for a genotoxicity test that models the initiation phase of tumor development as closely as possible in accordance with current carcinogenicity theory. The human–hamster hybrid A_L assay with embryonic chick cell cocultivation for metabolic activation models the initial mutation in human cancer as well or better than any other assay.^105,106 This contention is based on the following evidence: (1) Chemicals tested must pass through two intact biological membranes, that is, the CHO cell membrane and embryonic chick hepatocyte membrane; (2) rat liver S9 normally used for metabolic activation is itself mutagenic in the A_L assay, while embryonic chick cells provide an intact cellular metabolic activation system; (3) the mutagenicity target in the A_L assay is human chromosome 11; (4) mutagenicity is measured via loss of cell surface markers lost due to mutational damage at specific, identifiable places on human chromosome 11 thereby reducing measurement artifacts; and (5) mutations of any size from base changes to large chromosomal deletions are detected and quantified. The system can be automated with cell counting by multicolor flow cytometry.¹⁰⁷

Cigarette smoking is primarily a tumor promoter

Cigarette smoke is an extremely complex mixture (>4800 components¹⁰⁸) that contains small quantities of a large number of chemicals classified as IARC Group 1 (known human), IARC Group 2A (probable human), and IARC Group 2B (possible human) carcinogens.^108

–111 Concomitant with the presence of a large number of chemicals classifiable as IARC carcinogens, malignant lung tumors from smokers have been shown to possess different mutational profiles than lung tumors from nonsmokers.¹¹² Although less than 10% of smokers develop lung cancer,¹¹³ smoking causes between 80% and 90% of cases.¹¹⁴ The average age of smoking initiation is approximately 20, while the average age at diagnosis occurs some 50 years later at about age 70. Few cases are diagnosed in patients younger than age 45, with the majority of cases presenting at over age 65.¹¹⁵

The relatively older ages of lung cancer presentation are consistent with a sophisticated statistical analysis of the lung cancer epidemiological literature conducted by Flanders et al.¹¹⁶ These authors found that cigarette smoking duration is a stronger predictor of lung cancer mortality than is cigarette smoking intensity. This finding held regardless of age in both men and women. Lung cancer risk was proportional to approximately the second or third power of cigarette smoking duration among men and women 40–49 years of age. If tumor initiation were of greater importance, smoking intensity and its expected association with a greater number of initial mutations should be the predominant correlate of lung cancer risk. In contrast, the predominance of smoking duration is consistent with a promotional mechanism requiring a long period of time for the number of cellular divisions sufficient for development of a tumor (clonal expansion).

Estrogen can function as a tumor promoter

In the endometrium, three different estrogen-dependent pathways can function in tumor promotion: (a) Cellular proliferation can be stimulated by activation of the nuclear estrogen receptor-α leading to transcriptional activation of estrogen-responsive genes. (b) Activation of second messenger systems via estrogen binding to membrane-bound G-protein-coupled estrogen receptor. In cancer cells, estrogen enhances cellular proliferation via induction of extracellular signal-regulated kinase, phosphoinositide 3-kinase, and epidermal growth factor receptor. (c) The metabolic activation of estrogens produces free radicals which generate mutagenic DNA adducts. Accumulation of these adducts can transform proliferating cells to neoplasia.^{117

–122}

Development of endometrial cancer probably involves both genetic and epigenetic changes. Several different carcinogenic mechanisms have been identified including imbalance between endometrial proliferation via unopposed estrogen activity and the mismatch repair (MMR) system; hypermethylation of the MMR gene hMLH1; mutation of PTEN, β-catenin, and K-ras genes in type I endometrial cancer and of HER-2/neu and p53 genes in type II endometrial cancer; hypermethylation of SPRY2, RASSF1A, RSK4, CHFR, and CDH1; and methylation of tumor suppressor microRNAs, including miR-124, miR-126, miR-137, miR-491, miR-129-2, and miR-152. Oncogenic changes causing carcinogenesis include mutations and methylation of MMR genes. In addition, MMR mutation in germ cells and methylation patterns may be inherited over generations and cause familial tumorigenesis.¹²³

Breast cancer develops from the interaction of genetic factors as well as lifetime exposure to estrogen. Inherited BRCA1 and BRCA2 gene mutations are responsible for about 5–10% of breast cancers.¹²⁴ An important role for estrogen promotion is suggested by an extensive epidemiological literature. Women in Japan have a breast cancer rate much lower than that seen in women in the United States.^125,126 Japanese women who move to the United States experience rising breast cancer rates.¹²⁷ This migratory pattern of risk suggests that environmental factors can modulate the risk of breast cancer. Similarly, menarche among East Asian women ensues significantly later than in American teenage girls. Concomitantly, menopause occurs earlier in East Asian women than in American women leading to a lower lifetime exposure to estrogen stimulation of epithelial cells.¹²⁸ Breast cancer rates in East Asian women are lower than in American women due to the lower integrated lifetime dose of estrogen.

Promotion of prostate cancer

A high percentage of the prostates from elderly men contain small prostate cancers that will not become clinically significant during the patient’s remaining lifetime. Carter et al. reported that 50% of men between 70 and 80 years of age showed histological evidence of malignancy.¹²⁹ In 50-year-old men, the lifetime risk for developing histological evidence of prostate cancer is 42%.^129,130 However, in 50-year-old men, the risk of developing clinically significant disease is only 9.5%, with the risk of dying from prostate cancer even lower still at only 2.9%.¹³⁰

In 1987, Gardner and Culberson¹³¹ demonstrated that the conventional understanding of atrophic and proliferative changes as beginning in middle age or older was incorrect. A total of 51 prostates of men aged 19–29 years demonstrated a spectrum of proliferative abnormalities, including ductal and glandular hyperplasia, atypical hyperplasia, dysplasia, carcinoma in situ, and incipient adenocarcinoma. In addition, the majority of the prostates also contained substantial areas of atrophy. Patterns of atrophic change included cystic dilatation of glands with flattened epithelium apparently secondary to obstructive hyperplasia of ductal epithelium, areas comparable to sclerotic atrophy of the aged prostate and segments possessing the appearance of prepubertal unstimulated prostate. This study demonstrates that promotion of prostate cancer is a multi-decade process.

Obesity and tumor promotion

A large body of evidence from retrospective cohort studies suggests that obesity increases the risks of a variety of cancers¹³² occurring at the following sites: endometrial cancer,^133,134 esophageal adenocarcinoma,¹³⁵ gastric cardia cancer,¹³⁶ liver cancer,^137,138 kidney cancer,^139,140 multiple myeloma,¹⁴¹ meningioma,¹⁴² pancreatic cancer,¹⁴³ CRC,¹⁴⁴ gallbladder cancer,^145,146 breast cancer,^147

–150 ovarian cancer,¹⁵⁰ and thyroid cancer.¹⁵¹ The retrospective design of these studies cannot rule out the possibility of interpretative interference from confounding or colinearity, that is, whether there are unknown but causative differences not related to body weight between obese and thin individuals who presented with cancer.

Several mechanisms have been postulated to explain the epidemiological relationship between obesity and the risks of several different cancers. Obesity is associated with systemic chronic low-level inflammation.¹⁵² Inflammation can itself be mutagenic through the release of activated oxygen species but can also lead to increases in reparative cellular proliferation rates.¹⁰¹ In addition to systemic inflammation, obesity is also associated with several clinical conditions characterized by localized chronic inflammation.¹⁵³ Acid reflux from gastroesophageal reflux can lead to Barrett’s esophagus which is strongly associated with esophageal adenocarcinoma. Obesity is linked to gallstones and the attendant gallbladder inflammation is a risk factor for gallbladder cancer.¹⁵⁴ Chronic ulcerative colitis is a risk factor for colon cancer, and hepatitis is a risk factor for different types of liver cancer.¹⁵⁵ Adipose tissue produces estrogen, and obesity has been associated with increased risks of breast, endometrial, and ovarian cancers.¹⁵⁶

Obese patients frequently suffer from a prediabetic condition termed hyperinsulinemia or insulin resistance characterized by high blood levels of insulin or insulin-like growth factor-1 (IGF-1). Elevated insulin and IGF-1 may promote the growth of colon, kidney, prostate, and endometrial cancers.¹⁵⁷ Hormones termed adipokines are secreted by fat cells. The adipokine leptin promotes cellular proliferation. Blood levels of leptin increase with increasing body fat. Adiponectin, another adipokine, is less abundant in obese patients than in normal. Adiponectin displays antiproliferative effects. Fat cells may directly or indirectly effect cell growth regulators, for example, mammalian target of rapamycin and AMP-activated protein kinase. Obesity might also increase cancer risk by altering the three-dimensional mechanical properties of the cytoskeletal scaffolding surrounding breast cells thereby affecting signal transduction pathways and downstream DNA accessibility to transcriptional factors and thus gene expression.¹⁵⁸ Altered immune responses, adverse effects on the nuclear kappa beta system, and oxidative stress have also been invoked as possible mechanistic pathways by which obesity might increase breast cancer risk.¹⁵⁹

Atomic bomb survivors display lower cancer rates than would be expected if initiation were the primary carcinogenic mechanism

The various forms of ionizing radiation, that is, beta particles, alpha particles, and X-rays, can damage hydrogen bonds and base pairs in DNA, and cause single- and double-stranded breaks. Heavier charged alpha particles have a greater capacity to cause direct DNA damage while X-rays implement most of their DNA damage via indirect effects.^160
–162 In addition to DNA damage directly induced by ionizing radiation, highly reactive free radicals possessing unpaired electrons are also formed. These radiation-induced free radicals can damage membranes and other cellular structures that can alter cellular function or lead to cell death.^160
–162

Sustained exposure to radiation is a complete carcinogen as ionizing radiation is a mutagen and in the presence of continuing radiation exposure the mutated cells can undergo promotion and progression.^163,164 In contrast with chemical agents that distribute within cells based upon their lipophilicity,¹⁶⁵ charge,¹⁶⁵ and molecular size,¹⁶⁶ radiation penetrates cells and deposits energy within them in a random fashion. Radiation-induced cancers are of the same histological types as they occur spontaneously although the relative distribution of occurrence by organ site may differ. The risk of radiation-induced cancer can be modulated by noncarcinogenic secondary factors.¹⁶⁷

In August 1945, two atomic bombs were detonated over the Japanese cities of Hiroshima and Nagasaki with 5% of the total liberated energy in the form of ionizing radiation.¹⁶⁸ The enormous shock wave and extreme heat were the primary cause of deaths. By the end of 1945, approximately 140,000 people in Hiroshima and 74,000 in Nagasaki had died.^169,170

From the work of Preston et al.,^171,172 there were 848 excess solid cancers observed up to 1998 and 94 excess cases of leukemia up to 2000 in the exposed group according to radiation doses seen below in the two tables (Tables 7 and 8).¹⁷³ These data are from the Life Span Study (LSS) cohort that encompasses both Hiroshima and Nagasaki exposed populations.^168,174 The LSS population cohort of bomb survivors is split with 54,159 in Hiroshima (67.6%) and 26,002 in Nagasaki (32.4%).¹⁷⁵

Table 7.

Observed and excess solid cancers reported up to 1998 in the exposed group according to radiation dose.^a

Weighted colon dose (Gy)	LSS subjects	Observed cancers	Estimated excess solid cancers
0.005–0.1	27,789	4406	81
0.1–0.2	5527	968	75
0.2–0.5	5935	1144	179
0.5–1.0	3173	688	206
1.0–2.0	1647	460	196
>2.0	564	185	111
Total	44,635	7851	848

LSS: Life Span Study.

^aThis is a simplified version of table 9 in Preston et al.,¹⁷¹ which tabulates all cancers observed from 1958 through 1998 among LSS cohort members.

Table 8.

Observed and excess leukemia deaths reported up to 2000 in the exposed group according to radiation dose.^a

Weighted marrow dose (Gy)	LSS subjects	Observed cancers	Estimated excess solid cancers
0.005–0.1	30,387	69	4
0.1–0.2	5841	14	5
0.2–0.5	6304	27	10
0.5–1.0	3963	30	19
1.0–2.0	1972	39	28
>2.0	737	25	28
Total	49,204	204	94

LSS: Life Span Study.

^aThis is a simplified version of data in Preston et al.¹⁷²

Radiation is a mutagen (initiating agent). The atomic bomb survivors in Hiroshima and Nagasaki show relatively low cancer rates. These low cancer rates suggest that the initiating mutations, when not followed by promotional clonal expansion, do not sometimes result in development of cancer. The bomb survivor data are consistent with experimental results reported by Kiraly et al.¹ where mutations alone led to low levels of tumor development in rodents. In contrast, the same mutations in the presence of cellular proliferation displayed a high level of tumor development.

The effect of post-mutational events on the ultimate development of cancer in atomic bomb survivors was demonstrated by Sauvaget et al.¹⁷⁶ This group studied the effect of a diet rich in fruits and vegetables on modifying the long-term effects of radiation exposure on the clinical presentation of cancer. Radiation dose estimates were available for a cohort of 36,228 atomic bomb survivors of Hiroshima and Nagasaki. In 1980, these survivors for whom radiation dose estimates were currently available had their diet assessed and were then followed for a period of 20 years for cancer mortality. Using both an additive and multiplicative model for the joint effect of fruit and vegetable intake and radiation exposure on risk of cancer mortality, a daily intake of fruits and vegetables was associated with a significantly lower risk of cancer in atomic bomb survivors.

NTP studies do not appropriately evaluate genotoxicity and exhibit a high false-positive rate

Expert panels employed by NTP frequently use a weight-of-the-evidence approach to the genotoxicity evaluation of chemicals. This approach entails consideration of older historical genotoxicity results and those garnered from conduction of state-of-the-art genotoxicity assays conducted under good laboratory practices. This protocol introduces an unintended error rate into the genotoxicity evaluation process due to the poor quality of many historical genotoxicity assays.¹⁰⁰ In the NTP database, we previously identified 180 chemicals whose current genotoxicity test results are negative but that induce at least one tumor in either rats or mice.¹⁰⁰ The large number of NTP studies demonstrating at least one tumor in either rats or mice despite the particular chemical testing negative in the Ames Salmonella mutagenicity test supports the idea that mitogenesis due to cytotoxicity is inducing mutagenesis via amplification of the background mutation rate as described by Cohen and Ellwein,^177,178 Cohen et al.,¹⁷⁹ Moolgavkar and Knudson,¹⁸⁰ and Ames and Gold.¹⁸¹

Toward a more scientific process for assessing cancer risk of chemicals

Structural elements of carcinogenicity correlate strongly with ordinal ranks of tumorigenicity from 2-year NTP studies

In our previous analysis of the entire NTP database^{69,85,100

–104} and of the most commonly employed in silico techniques for the estimation of carcinogenicity from chemical structure,^85,104 we determined that structural elements of carcinogenicity strongly correlated with ordinal ranks of tumorigenicity.^85,104 Each of the 470 chemicals for which final technical reports were available reported results for male rats, female rats, male mice, and female mice. In several cases, one of the four studies on a particular sex/species category was deemed as “inadequate” due to technical problems with that arm of the study, while the three other arms reported valid results. This situation was amenable to statistical analysis with “inadequate” ranked just higher than “negative” due to the possibility that if that arm had been completed without technical difficulty, it might have shown a level of neoplasticity higher than “negative.” The descending order of categorical rank was as follows: Clear Evidence > Some Evidence > Equivocal Evidence > Inadequate Evidence > Negative Evidence. This ranking scheme resulted in a highest category of Clear (male rats), Clear (female rats), Clear (male mice), and Clear (female mice), and a lowest category of Negative (male rats), Negative (female rats), Negative (male mice), and Negative (female mice). Due to a sporadic presentation of species/sex categories ranked as “inadequate,” the final number of categories is not set at 48 as the size of the NTP database grows, but rather that is the number of categories that result given the outcomes from the 470 current chemicals for which there are final technical reports.

Analysis of the entire NTP database across all routes of administration consistently showed that the highest hurdle of neoplastic evidence was tumor site concordance across species.⁸⁵ This result created a boundary condition under which ordinal rank could be further split within neoplasticity category (1–48), but a chemical in a lower category could not be assigned a higher ordinal rank than that of any chemical in a higher category. The second highest hurdle of neoplastic evidence was tumor site concordance across sex within species. The final criterion influencing ordinal rank was multiplicity of tumors that were not concordant by organ site. These non-concordant tumors are referred to in the ranking scheme as “single tumors.”⁸⁵

In the work of Smith et al.,⁸⁵ the relationships between structural alerts of carcinogenesis, categorical rank (1–48) (Figure 1), and ordinal rank (1–135) were examined with the following results:

The Mann–Whitney–Wilcoxon rank sum test shows that the trend in structural alerts versus category ranking is highly significant (Z = -7:03; p value near 0); that is, positive structural alerts results are strongly associated with categorical ranks of increased tumorigenicity. The Mann–Whitney–Wilcoxon rank sum test shows that the trend in structural alerts versus ordinal ranking is highly significant (Z = -7:02; p value near 0). That is, positive structural alert results are strongly associated with ordinal ranks of increased tumorigenicity.

In the work of Smith and Perfetti,¹⁰¹ the sensitivity and specificity of using structural alerts of carcinogenicity to screen chemicals from the NTP database was noted as follows:

One hundred thirty-four of the 479 chemicals tested by way of inhalation, feed, gavage, drinking water, dermal administration, or intraperitoneal injection were negative [for tumor induction] in male and female rats and in male and female mice. Fifty-four of these 134 chemicals were ubiquitously negative for neoplasia; but nonetheless, contained a structural alert representing a false-positive rate of 40% (54/134). There were 330 chemicals that induced at least one tumor. Of these 330 chemicals, 54 chemicals did not possess a structural alert for carcinogenicity resulting in a false negative rate of 54/330 (16.4%).

The predictive power of structural alerts of carcinogenicity was superior to that seen when similarly employing the Oncologic program.¹⁰⁴ Overall, the false-positive and false-negative rates in terms of the ability of structural alerts of carcinogenicity to predict actual experimental results from the entire NTP database are fairly good. These results suggest that the current extremely expensive and time-consuming process of conducting 2-year rodent cancer bioassays is not providing results superior to the consideration of structural alerts of carcinogenicity. Recently, the United States Environmental Protection Agency embarked on a multiyear program to reduce or eliminate the use of laboratory animals.¹⁸² A similar effort is being undertaken by the pharmaceutical industry through the International Conference on Harmonization.¹⁸³

Noninvasive determination of pulmonary inflammation as a surrogate for tumor promotion potential

As discussed earlier in the text, initiated cells require clonal expansion (promotion) to eventually present as malignant tumors. Many known tumor-promoting agents or actions are beyond the ability of a workplace to prevent, for example, adverse effects from estrogen, testosterone, obesity, low intake of fruits and vegetables, and cigarette smoking. In general, the ongoing efforts to remove carcinogens from the workplace in developed countries have resulted in amelioration of exposure.¹⁸⁴ In general, workplace exposures from ingestion and dermal exposure are less common and easier to prevent than workplace exposure via inhalation as evidenced by the emphasis on inhalation exposures placed by the American Conference of Governmental and Industrial Hygienists (ACGIH) through issuance of Threshold Limit Value (TLV) recommendations.¹⁸⁴ The most preventable potential source of workplace tumor promotion is pulmonary inflammation. The general principle of tumor promotion via pulmonary inflammation in humans is probably best demonstrated by the large increase in lung cancer risk seen in cigarette smokers with chronic obstructive pulmonary disease (COPD) as compared with smokers at the same level of cigarette consumption who do not suffer from COPD.¹⁰⁰

The most cost-effective and high-quality method for assessing subclinical workplace inflammation is determination of the pro- and anti-inflammatory cytokine profile in expired breath condensate.¹⁸⁵ Workplace air monitoring can determine which areas of an industrial facility represent relatively low and high exposure zones. Workers from the low and high exposure zones who smoke cigarettes, have asthma, current lung infections, and so on would be excluded from study. Expired breath condensates can be collected via passive breathing for approximately 7 min. The collected condensates can be shipped to a specialized laboratory for analysis of a wide range of cytokines, for example, the University of North Carolina Cytokine and Biomarker Core Facility.¹⁸⁶

Conclusions

The vast majority of cancer deaths in the United States are due to invasive or metastasized carcinomas. Despite their relatively high cost and labor intensity, current tests for genotoxicity and tumorigenicity do not adequately model the risk of developing human carcinomas. Researchers from the Eleanor Roosevelt Cancer Institute, Colorado State University, and Dartmouth Medical School collaborated to develop an in vitro genotoxicity test that better addresses the mechanistic aspects of the initiation stage of tumor development, that is, the A_L human–hamster hybrid assay with embryonic chick cell metabolic activation. Unfortunately, this test is not currently employed for regulatory purposes. Structural elements of carcinogenicity estimate the rodent tumorigenicity of a chemical with an acceptable false-positive and false-negative rate of predictability. The tumor-promoting ability of inhalable chemicals can probably be ameliorated by lowering the exposure below the level that induces subclinical pulmonary inflammation as measured in expired breath condensate. Employing a combination of structural elements of carcinogenicity, results from the A_L assay, and noninvasive evaluations of pulmonary inflammation represent a more cost-effective, faster, and more mechanistically appropriate approach to the cancer hazard assessment of chemicals.

Footnotes

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

ORCID iD

Carr J Smith

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Rodent 2-year cancer bioassays and in vitro and in vivo genotoxicity tests insufficiently predict risk or model development of human carcinomas

Abstract

Keywords

Executive summary

Biggest cancer killers in the United States are carcinomas

Genetic and epigenetic basis of cancer development

Historical role of cancer bioassays

Two-stage mouse dermal promotion assay—initiation, promotion, progression

In humans, molecular stages analogous to tumor initiation, promotion, and progression are concomitant with the sequential histological stages of carcinoma development

Current genotoxicity testing protocols test for DNA damage analogous to tumor initiation rather than promotion (clonal expansion) or progression (carcinoma in situ advancing to invasive carcinoma or metastatic carcinoma)

Ames Salmonella mutagenicity assay

Chromosome aberration assay

MN assay

Comet assay

Big Blue TGR Mutation Assay

HGPRT mammalian mutation assay

Uniqueness of mutagenicity evaluation by the human–hamster hybrid AL assay with embryonic chick cell cocultivation for metabolic activation

Cigarette smoking is primarily a tumor promoter

Estrogen can function as a tumor promoter

Promotion of prostate cancer

Obesity and tumor promotion

Atomic bomb survivors display lower cancer rates than would be expected if initiation were the primary carcinogenic mechanism

NTP studies do not appropriately evaluate genotoxicity and exhibit a high false-positive rate

Toward a more scientific process for assessing cancer risk of chemicals

Structural elements of carcinogenicity correlate strongly with ordinal ranks of tumorigenicity from 2-year NTP studies

Noninvasive determination of pulmonary inflammation as a surrogate for tumor promotion potential

Conclusions

Footnotes

Declaration of conflicting interests

Funding

ORCID iD

References

Uniqueness of mutagenicity evaluation by the human–hamster hybrid A_L assay with embryonic chick cell cocultivation for metabolic activation