There is now widespread realization that many if not most cancers,
perhaps 80%, are caused by substances in the environment: cigarettes,
fossil fuels, pesticides, drugs, preservatives, food colors, dyes,
radiation, asbestos and many others.
Some of this has become known through the detection of cancer among people who are particularly exposed to these substances, including those engaged in their manufacture. As long ago as the 18th century, it was realized that the scrotal cancers of chimney sweeps were caused by skin irritation from soot. Investigations in recent decades have demonstrated that cancer can be induced in animals by an increasing number of these substances, including all those which have been proved to be carcinogenic in humans. But it is not known how many carcinogenic substances in animals will produce cancer in humans, nor can it be assumed that different species of animals are uniformly subject to cancer induction.
The different susceptibility of species is only the beginning
of an almost endless list of factors which affect an individual
animal's chance of acquiring the disease, and which influence
its nature and duration. Among these are sex, in-breeding, age,
diet, the combined effect of two or more carcinogens (may be additive
or inhibitory), husbandry (impurities in the diet, water or air,
bedding, housing conditions -single or group caging - crowding
stress, etc.), immune status, and the way the carcinogen is metabolized
in a particular species. This variability is further complicated
by a variation in response to each type of carcinogen: thus the
rabbit is refractory to liver tumor induction from azo compounds
but susceptible to skin tumors from polycyclic hydrocarbons, whereas
the rat is susceptible to these in opposite fashion. (Grice, H.,
Proceeding to the matter of the substance to be tested and the selection of dose and frequency of exposure, another Pandora's box of variability opens. Assuming that the objective is to extrapolate (transfer) to humans the cancer potential demonstrated in animals, there must be a relationship between the conditions under which both humans and animals are exposed to the substance being tested. Saccharin, for instance, fed in enormous doses to rats, produced bladder cancer, yet this hardly mimics the typical human exposure to comparatively minute quantities of the substance, especially when it is likely that such massive doses may overwhelm the natural defense mechanisms of the body, which normally can be counted on to defend against or even repair potentially cancerous changes in the genetic material of the cells. (Gehring, P., 1973).
Another problem is the frequency and duration of exposure to the test chemical. In order to be comparable to the very long development period of most cancer, the material may have to be administered daily over the life span of the test animal - and cancers are also very slow to develop in animals: in monkeys, the species closest to man, up to 10 years is required to develop tumors, and 5 to 10 years is not uncommon in dogs. Thus the investigator is forced to concentrate on rodents, but even with these a year or two may be required, during which all the conditions which may affect the experiment must be kept constant, and, if there are results worth reporting, these are still in species far removed from our own.
Finally, the impurity of the test substance can skew the results. With the increasing sensitivity of procedures such as the Ames Salmonella Test, a mutagenic (or carcinogenic) effect can be demonstrated in what was supposed to be a harmless substance simply because it was contaminated by a tiny amount of a potent carcinogen. In fact the saccharin in the experiment described above was demonstrated by the Salmonella test to contain traces of just such a substance, which might be another explanation for the cancers which appeared in the rats. (Donahue, E., 1978). The Grice study noted that "factors such as batch to batch variation, chemical synthetic processes, packaging, handling, storage and processing into food and drug preparations and interacting with other chemicals can have profound influences on the toxicity of the test material. Long-term studies should not be undertaken until the above information is available and unequivocally established." (Grice, H.,1975, p.20).
Unfortunately, the emphasis on the use of animals in bioassay of slow-developing cancer has resulted in vast numbers of such studies, all subject to the variable factors described above, and therefore frequently of questionable scientific value. Furthermore, if the variables were uncontrolled, or unrecognized and not reported, it is impossible for a later investigator to reproduce the experimental results. It is disturbing to think of the wastage of animals in these investigations, the loss of human time, and the expense.
Nevertheless, the high cost of carcinogen investigation in animals - an experiment involving typically 600 to 700 animals and costing more than $100,000 (Sontag, J., 1976) - and the disappointing results in terms of prevention or cure of the disease in humans, have raised a clamor for a fresh approach. Humane considerations have had little to do with it, but humanitarians can nevertheless find encouragement in the new trend.
In the light of recent developments in cancer research and
better understanding of the molecular and cellular mechanisms
involved, it has become retrospectively clear why the animal testing
of the past came up with so few clues to the nature of the disease
in humans. A brief review of the mechanism of carcinogenesis as
now understood will help to explain this.
It is now believed that the chemicals which act as carcinogens must be activated or potentiated in the body by enzymes which turn them into "electrophiles" - compounds which "love" electrons and which seek them out within the cell nuclei from DNA, which carries the genetic information for all cells, and RNA which transmits information from DNA to the protein-forming system of the cell. In the process of raiding DNA and RNA for electrons, the electrophiles become bound to DNA and cause a change or "mutation" in its genes. The genetic message is disrupted, and the result may be the initiation of an abnormal or cancer cell. Next, a second substance, or "promoter," which may be a hormone, causes the initiated cells to clump or pile up and grow chaotically into a cancerous tumor. The enzymes which help to start all this trouble can vary in different individuals, and even more so between species, thus explaining why humans differ one from another in susceptibility to cancer and, by the same token, differ even more in this respect from other species.
Obviously, if confusing variables increase when the eye of the scientist strays from human material, it behooves him to look to his own kind for some of the answers. Here are some of the techniques which are being developed.
Since experimental cancers cannot be safely initiated in human
beings, investigators have turned to the study of human cells,
tissues and even minute parts of organs which can be kept alive
in the laboratory if nourished by a suitable medium. "In
vitro" studies of animal tissues have also played their part;
indeed in certain circumstances are still valuable, but for the
reasons mentioned above the hope of the future seems to lie more
with the techniques which use human material to understand humans.
One of the breakthroughs occurred with the development of cultures
of human embryonic lung cells by Leonard Hayflick, reported in
1961. (Hayflick, L.,1961). These "WI 38" cells are able
to produce forty generations before dying. However, the cells
are fibroblasts, which differentiate into the connective tissue
of the body (tendons and the like), whereas most cancers are derived
from epithelial cells. Much effort has gone into attempts to culture
epithelial cells from various organs, also to grow and keep alive
for longer periods of time cells which were not merely embryonic
but more differentiated.
Important advances along these lines were discussed at a workshop at the Given Institute of Pathobiology, Aspen, Colorado, in Aug. 1977 sponsored by the Carcinogenesis Program of the National Cancer Institute. (Harris, C., 1978). Some of the reports of the symposium are summarized in the following paragraphs.
The secret of long life for cells in vitro has been found in the addition of specific substances to the culture medium, replacing as much as possible the traditional serum whose large protein content interfered with growth and which was subject to deterioration. Thus the addition of "epidermal growth factor" has tripled the culture life of epithelial keratinocytes from foreskins of newborn infants; cultured human mammary epithelial cells have been maintained with a mixture of hormones (estradiol, progesterone and prolactin), and it is now thought that each epithelial cell type may require a different mixture of hormones.
Significant advances in organ cultures or explants were discussed at the Given Workshop. It was reported:
"Bronchus, pancreatic duct, bladder, uterine cervix, breast, colon, esophagus and aorta can all be cultured from weeks to months with maintenance of normal-appearing epithelium. Chemically defined culture media have been developed for human bronchus, colon and pancreatic duct, which eliminates the biological variability due to serum from studies of carcinogen interactions in these tissues."
A major topic at the Given Workshop was the metabolism of chemical
carcinogens by human cells and tissues. The inter-individual variation
in binding levels of the carcinogenic polynuclear aromatic hydrocarbon
BP in liver biopsy specimens varied 30-fold, in cultured human
colon and bronchus 60 to 70-fold. These variations in an individual's
"metabolic profile" may be influenced by genetic factors,
disease states and the environment. Eventually, as Dr. U. Saffioti
suggested at a New York Academy of Sciences meeting in June 1978,
tests on biopsy material from individuals may help identify those
with particular susceptibility to certain carcinogens and warn
them to avoid them (e.g. cigarettes). Similarly, they might enable
an industry manufacturer using a potentially hazardous chemical
to exclude susceptible individuals, identified by an appropriate
test, from the workplace. (Edelson, E.,1978).
In addition to reports on the metabolism and DNA binding of known carcinogenic chemicals by cultures of human bronchus, colon and arteries, successful transformation of normal human cells from the mouth (fibroblasts) and foreskin into cancer cells was described. These last-mentioned experiments are similar to others (not reported at the Given Workshop) in which A.E. Freeman and others (Freeman, A., 1977) described a chemically induced cancer-like change in human skin cultures, and R. Tayler and D.W. Piper (Tayler, R., 1977) reported that cigarette smoke condensate produced a result on the stomach mucosal cells in organ culture typical of malignancy that did not differ from that of the known gastric carcinogen N-methyl-N'-nitro-N- nitrosoguanidine. With this research we are approaching a very significant stage in alternatives to cancer testing in animals: namely, the ability to produce, in the laboratory, cancers in human organs in response to chemical substances known to be mutagenic or carcinogenic.
The knowledge that a substance is "mutagenic " (capable
of producing a heritable change in the genetic material of a cell)
does not have to come from trial and error testing of innumerable
environmental chemicals in delicate cell or organ culture systems.
It is very cheaply and quickly demonstrable (for about $200 and
in 3 days for each assay) by the Ames test, using Salmonella
bacteria which cannot grow because a mutation has made them
unable to manufacture the amino acid histidine, a necessary nutrient.
The potential carcinogen to be tested is exposed to rat or human
liver extract and, just as would happen in the mammalian body,
is converted into metabolites. These metabolites, if they prove
mutagenic, will damage the DNA of the histidine deficient Salmonella
and cause some of them to revert back to their original "wild"
state. Once again able to manufacture histidine, the bacteria
grow into visible colonies whose number provide a quantitative
estimate of the mutagenic potency of the suspected carcinogen.
(McCann, J., 1976).
There are other fast, short-term tests for mutagenicity (under these conditions always related to carcinogenicity) which can be used to screen potential carcinogens and which do not involve animal suffering. A reliable one, known as the Cell Transformation test, uses a culture of neonatal Syrian hamster kidney fibroblasts. Interestingly, the LD/50 is calculated as part of the procedure, but here the "lethal dose" which kills 50% only slaughters cells, not whole animals as in its cruel application in toxicology. (Styles, J.,1977).
Since we live in an age increasingly dependent on chemicals
in innumerable forms to sustain our way of life, and since we
have become wary of them as potential carcinogens, a mandate to
test has been handed by Congress to the federal agencies concerned.
The National Cancer Institute's survey reports 7,000 chemicals
tested by long-term animal bioassay up to 1975, of which from
600 to 1,000 are potential carcinogens. But many of these were
selected for testing because they have a molecular structure similar
to known carcinogens; the number of chemicals actually producing
cancer in animals was estimated to be 221 in an international
survey (1978), and only 26 chemical substances or processes have
been implicated in cancer induction in humans (6 by animal tests
and 20 from epidemiological evidence). What is suspected but not
yet fully explored is the nature of so-called promoting
or potentiating agents which are themselves not carcinogenic
but which contribute to the development of cancer in subjects
who have been exposed to the actual carcinogens. (IRLG,1979, p.12).
Priorities of testing in this vast area must be established, and one method is by selecting compounds whose molecular structure, as mentioned above, resembles that of known carcinogens. (Ibid., p. 67). For instance, the polynuclear aromatic systems, and N-nitroso groups, are arrangements of atoms known to have carcinogenic properties. The more this kind of information can be developed through molecular analysis, the less will empirical screening of thousands of chemicals via animals be necessary - at least in the preliminary identification of potential carcinogens.
Very suggestive data on carcinogens often come from observations
described as "epidemiological." A well-known finding
is that the incidence of cancer of the lung in women has risen
proportionate to the increase in cigarette smoking in that group.
Another is the observation of cancer incidence and mortality difference
in people on a geographical basis -often confirmed by an altered
incidence in such a population after migration. And clinical case
reports may give early warning of a potential carcinogen. (Nat'l.
Cancer Advisory Bd.,1976, p.4).
But the incidence of cancer in workers exposed to chemicals, or toxic reactions, is particularly diagnostic, and should be continually monitored. This monitoring should not be confined to the workplace; it should be continued after workers have retired and, whenever feasible, studied in necropsy material after their death. This would throw light not merely on mortality and the more obvious morbidity, but also on the effects of low-level exposure and on time trends - some cancers, for instance, may not become manifest for several decades. (Anon., 1973).
The problem is how to instigate such studies, which would both spare animals and benefit humans. Industry may well have an ambivalent attitude toward epidemiological investigations. While it should be obvious that such studies are the best way to bring the hazards to workers and to those living near the factory into focus, and to suggest preventive measures, there has been fear that disclosure of toxic effects may lead to damage suits and crippling injunctions. Even the government has indulged in a cover-up of leukemia morbidity from nuclear exposure.
When monitoring has been attempted, it has probably relied on animal toxicity testing, with LD/50, behavioral studies and the like. The trouble with animal testing, aside from the inhumanity, and the difficulty in comparing animals and man, is that animals are subject to variables. For instance, as K.Z. Morgan points out, sensitivity to leukemia induction [from radiation], and life expectation, varies greatly with different kinds of mice. On the other hand, if one attempts to reduce the variables by developing inbred, carefully controlled strains, such animals are even less comparable to the human, who is a "wild or heterogeneous animal living in many types of environment with various eating and drug habits, with many diseases and eccentricities, of various ages and so on." (Morgan, K., 1979).
These variables in humans explain why one man may be vulnerable to cancer and another not, but the epidemiological approach, although it can predict how many per thousand are likely to develop cancer following exposure to certain known carcinogens, cannot readily pick out the individuals who will succumb to the disease. It works by hindsight, but what is also needed are tests that can be applied to individuals to detect early changes in the genetic material - DNA and RNA - of cells.
It has been suggested that a test which does correlate with
threatened cancer is a high chromosome breakage rate. This has
been found in workers exposed to benzene, a carcinogen and a highly
toxic chemical. However, critics of some recent work along these
lines have pointed out that variability, the ghost which haunts
so many predictive tests, is also present here to a degree which
makes the forecast of cancer in any individual very uncertain.
Variables which are said to affect chromosome breakage, according
to Dr. J.R. Venable, director of biochemical research for Dow
Chemical Co., are factors such as "the viruses workers might
have had, medication they might have been using, laboratory test
techniques and even the season of the year." The clincher
for carcinogenicity is, of course, the clinical appearance of
the disease, but the fact that this may not show itself for many
years has so far prevented confirmation, at least in the Dow study,
of the benzene-chromosome breakage-cancer linkage. (Severo, R.,
When a single test is faulted because of variability, lack of controls, or abbreviated follow-up, it simply means that a broader spectrum of tests has to be administered if a "metabolic profile" known to be characteristic of the cancer-prone is to emerge.
One such test has recently been reported by Lars Ehrenburg and his colleagues at the University of Stockholm. It bypasses animal testing and requires merely a small sample of blood from the person exposed to the toxic substance. It has been found that the amino acids in the hemoglobin of the blood, cysteine and histidine, react with vinylchloride and other alkylating agents suspected of inducing cancer, at a rate which can be simply related to that in DNA uptake, so this uptake in turn can be easily calculated. Since hemoglobin cells have an average life of about four months in the blood stream, a sample records exposure over about two months. A report states:
"So far statistics are too few to determine whether the method can predict cancers in people exposed to alkylating agents. But it can pick out those who have accidentally received larger than normal doses. As one of the fundamentals of cancer treatment is to catch the disease early, high hemoglobin alkylation could be a valuable indication that the individual should undergo further observation." (Anon., 1979e).
Another technique which shows increasing promise of being able
to sort out the numerous variables which occur in the metabolic
profile of an individual employs the mass spectrometer. An instrument
that can identify and quantify some 150 components of urine alone
may be extremely useful in "profiling" persons with
different types and stages of cancer and identifying the metabolic
abnormalities characteristic of their disease. Besides urine,
both blood serum and spinal fluid can be analyzed with no harm
to the patient. Epidemiological studies by this method are still
in the future, but may eventually be done on those who work with
toxic materials. This kind of investigation also bypasses animals
completely, and can be performed on humans with no ethical objection.
To repeat: more accurate cancer prediction is not achieved by testing a greater variety of animals but by exposing humans to a greater variety of tests. To borrow an analogy from navigation, by taking only one bearing you cannot determine where you are at sea. But if you take bearings on two or more objects, and note where the lines cross, you have your position.