The breadth of the ten key characteristics of human carcinogens

The International Agency for Research on Cancer (IARC) is employing a protocol for the initial evaluation of the carcinogenic potential of chemicals termed the ten key characteristics of human carcinogens (TKCs) (Smith et al. 2016; Bus 2017; Trosko 2017). The TKCs characterize an agent as follows: (1) Is electrophilic or can be metabolically activated; (2) Is genotoxic; (3) Alters DNA repair or causes genomic instability; (4) Induces epigenetic alterations; (5) Induces oxidative stress; (6) Induces chronic inflammation; (7) Is immunosuppressive; (8) Modulates receptor-mediated effects; (9) Causes immortalization; 10) Alters cell proliferation, cell death, or nutrient supply. It is striking that neither IARC nor the Smith et al. paper (2016) overtly mentions the problem of species-specific xenobiotic metabolism.

Given the high doses routinely applied in toxicology studies (Buckley and Dorato 2009; Smith and Perfetti 2020), tens of thousands of chemicals and pharmaceuticals currently in commerce would be expected to display a positive finding in one or more of the TKCs (IARC 2017). This is especially true in light of the fact that many in vivo studies employ a non-physiological route of administration (e.g., intraperitoneal).

Protocols for cancer hazard assessment of chemicals represent older technologies

Many of the procedures underlying the standard protocols for the assessment of the carcinogenic potential of chemicals were developed many decades ago. The rodent cancer bioassay utilizing mice and rats was developed in the 1960s (Cohen 2010a, b). Ames and his colleagues invented the eponymous Ames Test in the early 1970s (Ames et al. 1972; 1973a, b; McCann et al. 1975). While chromosome aberrations had been observed for over 100 years, Moorhead et al. developed the in vitro chromosome aberrations test in 1960 (Evan 1976; Natarajan 2002). In 1970, Boller and Schmid developed a test method to evaluate the frequency of micronucleated erythrocytes found among normal erythrocytes. This initial method used bone marrow and peripheral blood cells from the Chinese hamster following treatment with the strong alkylating agent, trenimon. Boller and Schmid (1970) termed this method the “Mikrokern-Test (micronucleus test)”. During the early to mid-1970's Schmid (1975) and Heddle's group (1973) further developed the protocols for conducting the micronucleus test.

In contrast with the older technologies currently employed in the assessment of the carcinogenic potential of chemicals, clinical oncology is leveraging an ever-improving understanding of the pathogenesis of cancer to bring new technologies to patients.

Increased mechanistic specificity is driving clinical oncology

Historically, once a cancer grew to a size sufficient to produce symptoms (Gospodarowicz et al. 1998; American Joint Committee on Cancer 2017), or intentional (Greene 2016) or serendipitous screening detected a tumor, it was staged thus defining its clinical extent (American Joint Committee on Cancer 2017) and graded for cellular changes related to the degree of differentiation (Horne 1992; Liu and Wu 2020). Many professional bodies, including the College of American Pathologists (CAP.org), have often internationally agreed to Cancer Protocols for reporting cancers (biopsies, resections). They include up-to-date requirements including additional testing that is needed for each type of cancer (Renshaw et al. 2018). As mechanistic understanding of human cancers progressed, an additional step of testing for the presence of specific genetic changes, found in only a subset of the patients presenting with that tumor type, could be conducted (USDHHS 2021). The presence or absence of these additional genetic markers guided the oncologist in the selection of the chemotherapy, immunotherapy, or surgical protocol with the highest probability of efficacy for the patient with that particular gross histological and molecular profile (CDC 2019). The tremendous activity directed toward the identification of specific molecular alterations in human cancers can be seen in the large number of relatively new cancer drugs targeting specific mutations or pathways that have been brought to market in the last 20 years (Table 1, Supplemental Materials).

Cancer hazard assessment of new chemicals should strive for specificity

Genetic changes are the foundation for the development of cancer, with multiple errors in a single cell necessary for cancer development (Shen and Laird 2013). These genetic changes can be inherited, can occur due to direct DNA damage (DNA reactivity), or secondary to spontaneous errors occurring during normal DNA replication. The degree of correlation between the genotoxicity test results or rodent tumor data and human cancer risk cannot be determined (Berry 2017; Smith et al. 2019). The main reason these in vitro genotoxicity tests can be misleading is because of the many assumptions made in interpreting positive results and built-in technical limitations of each assay. Specifically, in some of these assays, the presence or absence of a cellular protein for the gene can be due to a mutation or to an alteration in the expression of that gene (Trosko 2010; Trosko and Upham, 2010). Another concept that is ignored in a genotoxicity assay is that not all mutations are due to DNA damage that persists unrepaired, as in the skin-cancer prone human hereditary syndromes of xeroderma pigmentosum. In other words, while feeble genomic DNA repair after exposure to ultraviolet light is the very best example that mutations are correlated with repair defects, in all likelihood, errors of DNA replication of normal genomic DNA are the cause of most mutations in human cells (Loeb 2011; Tomasetti et al. 2017).

A further major source of contention is, what are the target cells that are altered and then progress to become the ultimate “cancer stem cells”? Human organ-specific adult stem cells exist in almost all if not all organs (Reya, 2001; Visvader and Lindeman 2012; Kreso and Dick 2014; Dawood et al. 2014; NIH 2016; Kuşoğlu and Biray Avcı 2019). These organ-specific stem cells have few mitochondria, whereas their terminally differentiated offspring contain many (Payandeh et al. 2020). Stem cells metabolize via glycolysis and their differentiated daughters metabolize glucose via oxidative phosphorylation (Shyh-Chang et al. 2013). This is also why in vivo detection of DNA adducts is sometimes misleading because they can reflect a chemical’s having damaged mitochondrial DNA in a progenitor (finite limited cell division cell) and terminally differentiated cells (Vitale et al. 2017). It has been proposed that the progenitor cells are not going to give rise to cancer (Trosko 2009).

This also raises a challenge to the idea that one of the characteristics of a carcinogen is its ability to induce immortalization. Conceptually, a stem cell is naturally immortal until it is induced by epigenetic mechanisms to differentiate or become mortal. Initiation is best described as the first step in the multi-step, multi-mechanism process of carcinogenesis. A normal, naturally immortal stem cell is unable to remain immortal because, during its differentiation, some event is blocked in an essentially irreversible manner. However as a result of an ‘initiation event,’ it can now clonally multiply without terminal differentiation (Trosko 2018).

An illustrative example of the attempt to better model the early mutational events in cancer pathogenesis was the collaborative development by the Eleanor Roosevelt Institute for Cancer Research, Colorado State University, and Geisel School of Medicine at Dartmouth of the human–hamster hybrid CHO A(L) assay (AL assay) with embryonic chick cell co-cultivation for metabolic activation (Collins et al. 1982; Waldren, 1983; Preston et al., 1991; Waldren et al. 1999; Gustafson et al. 2000). The Chinese hamster ovary cell line CHO A(L) that stably incorporates human chromosome 11 is described by Ross et al. (2007).

The AL assay models the initial mutation in human cancer in the following ways: (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 AL assay, while embryonic chick cells provide an intact cellular metabolic activation system; (3) the mutagenicity target in the AL 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 (McNiel 2000a, b; Zhou et al. 2006). While pointing in the correct conceptual direction, the AL assay is not practical as a screening tool for large numbers of chemicals because it is labor-intensive and relatively expensive.

While the degree of mechanistic specificity in chemical hazard assessment screening protocols would not be expected to reach the level seen in clinical oncology (Table 1, Supplemental Materials), an increased appreciation of the role played by particular Modes of Action (MOA) in the induction of certain important carcinomas is needed (Cohen et al. 2020). Examples of such work are the publications of the International Programme of Chemical Safety (IPCS) MOA framework in 2001 and the development by the International Life Sciences Institute Risk Science Institute (ILSI RSI) of a Human Cancer Relevance Framework. IPCS has combined and extended these components to produce a unified Human Cancer Relevance Framework (IPCS HRF) (Sonich-Mullin et al. 2001; Meek et al. 2003; Seed et al., 2005; Boobis et al. 2006; 2008).

The particularly important role played by post-initiating events in the development of human carcinomas is demonstrated by the case of the archetypal initiating agent, i.e., ionizing radiation. Japanese atomic bomb survivors have been followed in the Life Span Study (LSS) for decades. Despite being exposed to a strong burst of ionizing radiation expected to induce a significant number of mutations, of 22,538 incidences of first primary solid cancer cases identified in the LSS population, only 4.4% (992/22,538) * 100 = 4.4%) was associated with radiation exposure (Grant et al. 2017) (Fig. 1). In a previous report on this same LSS population, about 440 of 9335 of the deaths due to solid cancers (4.7%) were attributed to radiation exposure (Preston et al. 2003). Therefore, radiation exposure is associated with 4 – 5% of cancer incidence and death (Preston et al. 2003; Grant et al. 2015, 2017). This result suggests that factors other than ionizing radiation-induction of point mutations are playing an important role in the development of human cancers. One possibility is a role for caloric restriction, and the Japanese diet, in the modification of risks for cancer before and shortly after the atomic bomb exposures. Caloric restriction is a well-known reducer of many chronic diseases (Golbidi et al. 2017; Rizza et al. 2013; Anton and Leeuwenburgh 2013; Omodei and Fontana 2011; Dirks and Leeuwenburgh 2006).

Fig. 1
figure 1

Incidence of cases of solid cancers in Japanese atomic bomb survivors 1958–2009

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The unique Japanese diet, before and shortly after the acute exposures to radiation, was characterized by a low caloric level, reliance upon vegetables, raw fish, soy products, green tea, and rice, with no meat. Keeping in mind, historically at that time, the stature and breast cancer frequency in Japanese women was very low. Today, because of the shift in the Japanese diet toward a more Western diet, the frequency of breast cancer in Japanese women is approaching that of the frequencies of Western women (Hirose et al. 2003).

The ten key characteristics of cancer as viewed by IARC and the new hallmarks of cancer (NHC) by molecular oncologist

If one is to try to identify characteristics of an agent (microbiological, physical, chemical, etc.) that might contribute to the multi-stage, multi-mechanism pathogenesis of cancer, then one must not only seek specificity in one set (e.g., the TKCs), but also with others (e.g., the NHC), in the end, different conceptualizations should be compatible with one another as to consistency and plausibility. Given the criticisms regarding some characteristics of the TKCs, examinations of the misleading Hallmarks must be treated similarly. In their updated Hallmarks of Cancer, Hanahan and Weinberg, (2011) conceptualized these characteristics.

Examination of the characteristics of the hallmarks of cancer demonstrates that they are not unique to cancer cells, but can be intrinsic to normal adult organ-specific stem cells.

Hanahan and Weinberg (2011) have provided evidence for the stem cell hypothesis of cancer and that the normal, immortal adult stem cell, once blocked from terminal differentiation remains immortal and is not induced to mortality. Normal stem cells are resistant to agents that can damage genomic DNA (Vitale et al. 2017). These normal adult stem cells can invade other tissues/organs during development, as cancer stem cells. These normal adult organ-specific stem cells also can induce angiogenesis. Apparently, these adult organ-specific stem cells are susceptible to either differentiation or apoptosis-inducing agents (Vitale et al. 2017).

Knowing the Mode of Action of a non-DNA reactive chemical is extremely important. Only if one understands the total context of the user’s exposure, can a cautious decision be made. In all likelihood, the pharmaceutical industry has thrown out thousands of potentially efficacious drugs based on not taking into account this complex context.

Seeing the non-specifics in both of these attempts to provide generalized concepts for determining “carcinogenicity” of any agent associated with cancer appearing after exposure, calls for a reevaluation. Hanahan and Weinberg, have stated:

“Some would argue that the search for the origin and treatment of this disease will continue over the next quarter century in much the same manner as it has in the recent past, by adding further layers of complexity to the scientific literature that is already complex almost beyond measure. But we anticipate otherwise: those researching the cancer problem will be practicing a dramatically different type of science than we have experienced over the past 25 years. Surely much of this change will be apparent at the technical level. But ultimately, the more fundamental change will be conceptual. “(Hanahan and Weinberg, 2010.)

The determination of any agent associated with a cancer has to be viewed as to its mechanistic role as either (a) a mutagen (error of DNA repair of genomic DNA or error of DNA replication of normal adult organ-specific stem cells); (b) a cytotoxicant (a necrotic cytotoxicant, an apoptosis inducer or blocker, an autophagy-inducer, a cell removal via injury or surgery to cause compensatory hyperplasia or; (c) an epigenetic agent that inappropriately alters gene expression at threshold levels. The search for simple categorizations of agents that might contribute to the appearance of a cancer will likely not be fruitful.

Conclusions

The ten key characteristics of human cancer are too broad and non-specific to be useful as a protocol for evaluating the potential cancer hazard of chemicals. Extremely high false-positive rates might communicate to the public the false notion that everything is a carcinogen. As knowledge regarding the causation and progression of human cancers increases, this should be applied to the development of increasingly relevant testing protocols for the identification of potential human carcinogens, with particular emphasis on dose–response relationships.