Bernard W. Stewart

Key Characteristics of Carcinogens as a Basis for Organizing Data on Mechanisms of Carcinogenesis.

Background: A recent review by the International Agency for Research on Cancer (IARC) updated the assessments of the > 100 agents classified as Group 1, carcinogenic to humans (IARC Monographs Volume 100, parts A–F). This exercise was complicated by the absence of a broadly accepted, systematic method for evaluating mechanistic data to support conclusions regarding human hazard from exposure to carcinogens. Objectives and Methods: IARC therefore convened two workshops in which an international Working Group of experts identified 10 key characteristics, one or more of which are commonly exhibited by established human carcinogens. Discussion: These characteristics provide the basis for an objective approach to identifying and organizing results from pertinent mechanistic studies. The 10 characteristics are the abilities of an agent to 1) act as an electrophile either directly or after metabolic activation; 2) be genotoxic; 3) alter DNA repair or cause genomic instability; 4) induce epigenetic alterations; 5) induce oxidative stress; 6) induce chronic inflammation; 7) be immunosuppressive; 8) modulate receptor-mediated effects; 9) cause immortalization; and 10) alter cell proliferation, cell death, or nutrient supply. Conclusion: We describe the use of the 10 key characteristics to conduct a systematic literature search focused on relevant end points and construct a graphical representation of the identified mechanistic information. Next, we use benzene and polychlorinated biphenyls as examples to illustrate how this approach may work in practice. The approach described is similar in many respects to those currently being implemented by the U.S. EPA’s Integrated Risk Information System Program and the U.S. National Toxicology Program. Citation: Smith MT, Guyton KZ, Gibbons CF, Fritz JM, Portier CJ, Rusyn I, DeMarini DM, Caldwell JC, Kavlock RJ, Lambert P, Hecht SS, Bucher JR, Stewart BW, Baan R, Cogliano VJ, Straif K. 2016. Key characteristics of carcinogens as a basis for organizing data on mechanisms of carcinogenesis. Environ Health Perspect 124:713–721; http://dx.doi.org/10.1289/ehp.1509912

Involvement of MDR1 P-glycoprotein in multifactorial resistance to methotrexate

Cellular resistance to methotrexate (MTX) is believed to be unaffected by expression of MDRI P‐glycoprotein (Pgp), a pleiotropic efflux pump acting on different hydrophobic compounds that enter cells by passive diffusion. A series of human leukemic CCRF‐CEM sublines, isolated by multi‐step selection for very high resistance to MTX, exhibit multiple mechanisms of MTX resistance, including decreased carrier‐mediated uptake of MTX and DHFR gene amplification. These sublines show cross‐resistance to drugs of the multi‐drug resistance (MDR) family, which is correlated with relative resistance to MTX. The MTX‐selected sublines show increased expression and function of the MDRI gene, based on the measurement of MDRI mRNA, Pgp and rhodamine 123 accumulation. Sequence analysis of the MDRI cDNA from MTX‐selected CCRF‐CEM cells revealed no mutations in the protein coding region. MTX resistance in these cell lines is partially reversible by a Pgp‐specific monoclonal antibody (MAb) UIC2 and a monovalent Fab fragment of UIC2. Our results indicate that Pgp can contribute to multifactorial resistance to MTX.

Micrococcal nuclease: its specificity and use for chromatin analysis.

Micrococcal nuclease (MNase; EC 3.1.3 1.1) was first described by Cunningham et al. who, in 1956, recovered a deoxyribonuclease from culture supematant of the bacterium Micrococcuspyogenes var. aureus (now named Staphylococcus aureus). This extracellular nuclease was unusually thermostable, required calcium as a cationic activator and hydrolysed the 5’-phosphodiester bond of deoxyribonucleotides to yield 3’-monoand oligonucleotides and a free 5’-hydroxyl group. The enzyme is comprised of 159 amino acid residues and weighs 16,800 dalton (Taniuchi et al., 1967) and optimal nucleolytic activity is obtained at pH 8.6 with calcium ions at 10mM (Cunningham, 1958). Cunningham initially called the enzyme “micrococcal DNase”, however, he renamed it “micrococcal nuclease” due to its ribonuclease activity (Cunningham, 1958). The two strains of S. aureus used as a source of MNase are Foggi and V8, with the higher yielding Foggi strain used for commercial enzyme production (Anfinsen et al., 1971). The requirement for calcium cannot be met by magnesium ions, however, weak activity for DNA may be obtained with strontium ions, whereas activity is completely lost in the presence of EDTA (Heins et al., 1967; Cotton et al., 1979). The ribonuclease and deoxyribonuclease activities of MNase are competitively inhibited by a number of 5’-monophosphonucleotide derivatives, deoxythymidine 3’,5’diphosphate (pdTp; the most tightly binding inhibitor) and some heavy metal cations (Cotton et al., 1979). Analysis of the crystal structure of the pdTp-MNase complex has shown the DNA binding site to be a groove which binds three phosphate groups to the 3’ side of the hydrolyic site of a single DNA strand (Anfinsen et al., 1971). Further chemical properties of the enzyme have been reviewed by Anfinsen et al. (1971). The specificity of MNase for cleavage of DNA in the linker region between nucleosomes (discussed in

Designated “no smoking” areas provide from partial to no protection from environmental tobacco smoke

Objective: To determine the efficacy of designated “no smoking” areas in the hospitality industry as a means of providing protection from environmental tobacco smoke (ETS), and whether certain design features assist in achieving this end. Methodology: In the greater metropolitan region of Sydney, a representative group of 17 social and gaming clubs, licensed to serve alcoholic beverages and in which, apart from designated areas, smoking occurs, agreed to participate. In each establishment, simultaneous single measurements of atmospheric nicotine, particulate matter (10 μm; PM10) and carbon dioxide (CO2) levels were measured in a general use area and in a designated “no smoking” area during times of normal operation, together with the levels in outdoor air (PM10 and CO2 only). Analyses were made of these data to assess the extent to which persons using the “no smoking” areas were protected from exposure to ETS. Results: By comparison with levels in general use areas, nicotine and particulate matter levels were significantly less in the “no smoking” areas, but were still readily detectable at higher than ambient levels. For nicotine, mean (SD) levels were 100.5 (45.3) μg/m3 in the areas where smoking occurred and 41.3 (16.1) μg/m3 in the “no smoking” areas. Corresponding PM10 levels were 460 (196) μg/m3 and 210 (210) μg/m3, while outdoor levels were 61 (23) μg/m3. The reduction in pollutants achieved through a separate room being designated “no smoking” was only marginally better than the reduction achieved when a “no smoking” area was contiguous with a smoking area. CO2 levels were relatively uninformative. Conclusion: Provision of designated “no smoking” areas in licensed (gaming) clubs in New South Wales, Australia, provides, at best, partial protection from ETS—typically about a 50% reduction in exposure. The protection afforded is less than users might reasonably have understood and is not comparable with protection afforded by prohibiting smoking on the premises.

The prognostic value of MDR1 gene expression in primary untreated neuroblastoma

The contribution of MDR1 gene expression to the biology of childhood neuroblastoma is unclear. To clarify the role of MDR1 in this malignancy, we examined the relationship between MDR1 expression and patient outcome in subsets of 60 primary untreated neuroblastomas for which MYCN gene copy number and expression of the multidrug resistance-associated-protein (MRP) gene had been previously characterised. In contrast to MRP gene expression, MDR1 expression was lower in tumours with MYCN gene amplification compared with those without amplification. Strong correlations between MDR1 and MRP gene expression, and between MDR1 and MYCN gene expression, were observed in tumours lacking MYCN gene amplification (P < 0.0005). In these single-copy tumours, very high MDR1 gene expression was significantly associated with poor outcome (P < 0.05). Very high MDR1 expression was also strongly predictive of poor outcome in older children (P < 0.0001), but not in infants. These findings suggest a clinical role for the MDR1 gene in specific subgroups of primary neuroblastoma.

Cellular injury and carcinogenesis. Evidence for the alkylation of rat liver nucleic acids in vivo by N-nitrosomorpholine

An intraperitoneal dose of nitrosomorpholine (400 mg/kg body wt.) becomes rapidly distributed throughout the tissues of the rat. The concentration in the tissues falls to less than 10% of the initial value within 18 h of injection. After administration of the same dose of [3-14C] nitrosomorpholine 3.3% of the radioactivity was exhaled as 14CO2 over a period of 24 h and 81% excreted in the urine. 24% of the dose was excreted in the urine as unchanged nitrosomorpholine and 15% as a compound tentatively identified by its chromatographic behaviour as nitrosodiethanolamine. Evidence was found for the conversion in vivo of nitrosomorpholine to chemically active compounds capable of reacting with nucleic acids. An acid hydrolysate of RNA and DNA from the livers of rats given [3-14C] nitrosomorpholine contained six radioactive compounds which could be separated by chromatography on Dowex 50 as well as some radioactivity not retained on the column. From its chromatographic behaviour one of these six compounds appears to be 7-(2-hydroxyethyl)guanine. The others were not identified. There was no evidence for the formation of N-7-methylguanine. Eine i. p. verabreichte Dosis von Nitrosomorpholin (400 mg/kg Körpergewicht) wird rasch in allen Geweben der Ratte verteilt. Die Konzentration in den Geweben fällt innerhalb von 18 Std nach der Injektion auf weniger als 10% des Anfangswertes ab. Nach Verabreichung derselben Dosis von [3-14C]-Nitrosomorpholin wurden innerhalb von 24 Std 3,3% der Radioaktivität als 14CO2 ausgeatmet und 81% mit dem Urin ausgeschieden. 24% der Dosis wurden im Urin als unverändertes Nitrosomorpholin und 15% als eine Verbindung ausgeschieden, die vorläufig durch ihr chromatographisches Verhalten als Nitroso-diäthanolamin identifiziert wurde. Es wurden Anhaltspunkte dafür gefunden, daß Nitrosomorpholin in vivo in chemisch aktive Verbindungen umgewandelt wird, die in der Lage sind mit Nucleinsäuren zu reagieren. Ein Säurehydrolysat der RNA und DNA aus Leber von Ratten, denen [3-14C]-Nitrosomorpholin gegeben wurde, enthielt 6 radioaktive Verbindungen, die durch Chromatographie auf Dowex 50 getrennt werden konnten und außerdem etwas Radioaktivität, die nicht auf der Säule zurückgehalten wurde. Nach dem chromatographischen Verhalten scheint eine der 6 Verbindungen 7-(2-Hydroxyäthyl)guanin zu sein. Die anderen wurden nicht identifiziert. Es ergab sich kein Hinweis für die Bildung von N-7-methyl-guanin.

Differences in the carcinogenic evaluation of glyphosate between the International Agency for Research on Cancer (IARC) and the European Food Safety Authority (EFSA)

The International Agency for Research on Cancer (IARC) Monographs Programme identifies chemicals, drugs, mixtures, occupational exposures, lifestyles and personal habits, and physical and biological agents that cause cancer in humans and has evaluated about 1000 agents since 1971. Monographs are written by ad hoc Working Groups (WGs) of international scientific experts over a period of about 12 months ending in an eight-day meeting. The WG evaluates all of the publicly available scientific information on each substance and, through a transparent and rigorous process,1 decides on the degree to which the scientific evidence supports that substances potential to cause or not cause cancer in humans. For Monograph 112,2 17 expert scientists evaluated the carcinogenic hazard for four insecticides and the herbicide glyphosate.3 The WG concluded that the data for glyphosate meet the criteria for classification as a probable human carcinogen . The European Food Safety Authority (EFSA) is the primary agency of the European Union for risk assessments regarding food safety. In October 2015, EFSA reported4 on their evaluation of the Renewal Assessment Report5 (RAR) for glyphosate that was prepared by the Rapporteur Member State, the German Federal Institute for Risk Assessment (BfR). EFSA concluded that ‘glyphosate is unlikely to pose a carcinogenic hazard to humans and the evidence does not support classification with regard to its carcinogenic potential’. Addendum 1 (the BfR Addendum) of the RAR5 discusses the scientific rationale for differing from the IARC WG conclusion. Serious flaws in the scientific evaluation in the RAR incorrectly characterise the potential for a carcinogenic hazard from exposure to glyphosate. Since the RAR is the basis for the European Food Safety Agency (EFSA) conclusion,4 it is critical that these shortcomings are corrected. EFSA concluded ‘that there is very limited evidence for an association between glyphosate-based formulations …

P-glycoprotein-mediated methotrexate resistance in CCRF-CEM sublines deficient in methotrexate accumulation due to a point mutation in the reduced folate carrier gene.

We have previously described a series of methotrexate (MTX)‐selected CCRF‐CEM sublines (CEM/MTX R1–3) displaying increased resistance to drugs associated with the multidrug resistance phenotype and have provided evidence that MDR1 P‐glycoprotein contributes to multifactorial MTX resistance in these cells. We have also suggested that P‐glycoprotein‐mediated MTX transport arises in these cells due to a deficiency in the normal MTX transport route, the reduced folate carrier (RFC). We have now determined the nucleotide sequence of the RFC gene in CEM/MTX R1–3 cells and confirm that the carrier is defective in these cells as a result of a premature stop mutation at codon 99, which severely truncates the encoded protein. CEM/MTX R3 cells were removed from MTX, and a series of sublines with increasing MDR1 expression were derived, following selection with vincristine. These cells show increasing cross‐resistance to vincristine as well as other drugs associated with the multidrug resistance phenotype. More importantly, the increased P‐glycoprotein expression correlates with increased resistance to MTX, supporting the hypothesis that in cells with a defective carrier protein, MTX can become a substrate for P‐glycoprotein. Our data have implications for the P‐glycoprotein‐mediated transport of other hydrophilic drugs in situations where the relevant carrier protein has been functionally inhibited. Int. J. Cancer 78:176–181, 1998.© 1998 Wiley‐Liss, Inc.

Cancer Prevention: A Global Perspective

This review will be restricted to measures aimed at primary prevention of cancer; reduction of the death rate through screening will not be covered. In many instances, cancer prevention is just one outcome of the benefits of good health practices, which may also benefit cardiovascular, lung, infectious, and metabolic diseases. Thus, reducing tobacco smoking will bring benefits in heart disease, neonatal and maternal health, stroke, and peripheral vascular disease as well as a variety of cancers, while dietary advice appropriate to cancer risk reduction will bring benefits in diabetes, stroke, kidney, and heart disease.

Cancer prevention as part of precision medicine: “plenty to be done”

Summary No country can afford to treat its way out of the cancer problem. This review charts the increasing burden, summarizes the causes and describes opportunities for prevention and early detection, including precision prevention based on recent advances in cancer biology.