Invited Responses to Issues Associated with the Adaptive Response: Perspectives from Studies in Mammalian Cells on the Response to Low Radiation Doses or to Cisplatin

Kirsten A. Skov, Ph.D.

B.C. Cancer Research Centre

Advanced Therapeutics

601 W. 10th Ave.

Vancouver B.C. V5Z 1L3, CANADA

Tel: (604) 877-6098 x3021

Fax: (604) 877-6011

Email: kskov@bccancer.bc.ca

BACKGROUND

The term "adaptive response" means different things to different people. Some find stimulated growth of bacteria; others find turned on repair genes; some find protection against induction of mutations; and others describe "unusual" dose-response curves. In fact, there are at least three phenomena, listed below, which are considered in this issue of BELLE. These are responses to various naturally occurring and "man-made" insults, both chemical and physical. Such responses may vary from one organism to another, and indeed even from one cell type to another within a complex organism such as a mammal. Thus in fact there are many adaptive responses.

1. In the author's view, the singular term, adaptive response (AR), applies to the phenomenon of protection afforded by a small dose of radiation. This has been studied extensively by Wolf's group (1).*
2. Hormesis refers to a behaviour stimulated by a low level of insult, resulting in "improved" outcome# and has been described decades ago.
3. Another phenomenon which has been described only more recently in mammalian cells (4), exhibits non-linearity of response at low doses/concentrations and may be called "increased radioresistance", IRR, seen in many cell lines at low radiation doses (Figure 1).

Figure 1.
IRR (Increased Radioresistance) and HRS Hyperradiosensitivity), left panel; Priming (P) removes HRS (upper right panel) and Sensitizing (S) removes IRR, lower right.

This contribution to the five questions draws mainly on the latter. Reviews of this area e.g. (5-8) include comparison with the adaptive response to radiation (9) since small doses of damage prime cells to be more resistant (next paragraph). Hyper-radiosensitivity (HRS) is relevant to cancer treatment and possibly to cancer induction. Results on the drug cisplatin are included to illustrate such concepts using a toxic chemical - structure in the dose-response curve, and priming. Furthermore, there is usually more than one simple response to a given insult (heat, light, chemicals, radiation) and there seems to be some overlap between the families of responses. However, they are not identical.

Priming refers to resistance or protection turned on after a "tickle" dose of an insult which affects the response to the challenge dose. For AR or hormesis, this means better/higher/more improved endpoint. In the case of IRR, this protection results from removing the hyper-radiosensitive portion (HRS) ("P" in Figure 1 - prime or protect), e.g. (10). There certainly can be an overlap of challenge/responses as well - one insult can prime for another, as noted some time ago (11) - and there are many additional examples in the literature. Taking the anti-cancer agent cisplatin as one chemical example: interaction with other modalities such as Photo-dynamic therapy (PDT) and irradiation has been reported (6, 12, 13) which may eventually be linked to the well-known crossresistance e.g. (14, 15). Priming against cisplatin toxicity by UV-damaged DNA has been reported (16).

The common factors needed to turn on these responses are still under study. Radiobiologists assume initially that DNA damage is the trigger (but which type of damage: breaks, base damage or ? ), and consequently hypothesize that DNA repair must be the factor(s) turned on (4, 17). There is some evidence for this idea e.g. (18, 19), which would require protein synthesis, and indeed inhibitors do prevent IRR priming (6, 9, 10). Or, rather than extent of repair, perhaps fidelity of repair is improved. However, membrane damage has also been suggested to turn on some of these responses (8). Finally, and on the other hand: rather than turning on growth, turning off death (apoptosis) may be relevant (20). Whatever the mechanisms, we may ask whether these overlapping responses to insults are detrimental, or whether they can be exploited. For example:

· Is it possible that a worker exposed to low doses of radiation has less risk of cancer? (suggested by the results from the effects of radon (21) (22))
· Is it possible that diagnostic radiation could decrease cancer risk? (suggested by the fluoroscopy data (23) in (21)).
· Can a space traveler be "primed" by some chemical agent or nutritional factor (8) to better deal with the inevitable doses of various types of radiation?

We can attempt to learn from controlled systems - but how do we extrapolate to humans? Even if we knew the answers for averages, how would we ever apply this to human individuals with varying responses (genes), varying lifestyles and different environments? That there are genetic factors is clear.** However, we are not yet at the point of knowing all of the relevant aspects and how and whom to screen.

These are some of the issues which influence the following responses to the questions posed by the editor. It is also perhaps our challenge to eventually generalize, to explain all these behaviours in a unifying theory. Are each of these phenomena reflections of the initial overshoot (imbalance) in the feedback mechanism, caused by the additional stress over background, as described by Stebbing using control theory to explain hormesis (3, 24)? They are all responses to stress. Stress is a word used more and more, even by children and teens who are "stressed out". Yet if a person has no worries, he often creates them - the human seems to need problems and stress. One can ponder whether this extends somehow to physical and chemical stresses.

1. How does the dose that induces the adaptive response (AR) relate to human and environmental (ecological) exposures?

Some biological organisms exhibit cyclic behaviour (as a function of time) in their responses to endogenous insult (especially when compared to the unperturbed organism). This has been explained in terms of control and feed back mechanisms, the mathematics for which has been worked out many years ago for Control Theory (electrical circuits). Integration over time can yield results with "better" growth, "larger" colony size, etc. or hormesis - "by-products of growth control mechanisms due to regulatory over-corrections against the stress above background" (3, 24); perhaps this approach will be useful for other biological responses such as IRR/HRS and AR. Each organism contains enzymes and signal transducing features for homeostasis. Normally we are unable to measure oscillations directlyalthough examples do exist (3)(24)and instead we detect the integrated effects in an entire population. The amplitude after possible over-correction will depend on the size and timing of the insult, as well as on the type of organism, the sensitivity of the control factors, etc. and the background levels. A perturbation in the environmental exposure therefore could have results ranging from increased amplitude to damping of the oscillations, or no effect, given the large backgrounds (e.g. DNA damage equivalent to 50-100cGy per hour (25); see also discussion in (21)). Feedback responses need down- regulation (e.g. apoptosis) as well as up-regulation.

Thus, there is not one single answer to question 1. The type of insult, the organism, the timing, the sensitivity (control point), the magnitude of insult relative to amplitude, etc., would all be relevant. For humans, there are clearly well-documented doses in the environment which produce adaptation - an obvious example being alcohol tolerance. For individuals, genetics will also play a role (e.g. certain ethnic groups lack sufficient levels of enzymes which metabolize ethanol).

2. What are the potential up and down sides of having one's AR induced?

In terms of Control Theory, there are clearly tight feedback mechanisms without which we could not survive. ARs arise when there is some measurable perturbation, and hence they may merely be a manifestation of an imbalance in the system's response. In this framework, one can not really speak of upsides and downsides. In a general sense, we can say: There are many upsides - (a) we couldn't enjoy drinking beer without it! (b) environmental radiation (21, 22) or medical use of radiation (Fig. 6 in (21)) may protect against cancer (although there is a limit!). There may also be downsides - the inter-relatedness of the insults and responses means that an increase or decrease in amplitude could occur, depending on the timing and magnitude of each insult, which might interfere with a protective response. Also, in cancer treatment, a small dose of x-rays, which turns on IRR, could protect against a chemical treatment (drug therapy) (seen at least in vitro, (26)), or a chemical (drug) may turn on protection against radiation (therapy) (again in vitro (6, 26)).

3. Can the induction of AR be manipulated for medical and other benefits?

Continuing to use mainly the example of treatment of cancer by radiation and the response to low doses (Fig. 1) as an example (7), the answer is "probably". In cell lines at least, there seems to be a correlation between the intrinsic radiosensitivity (SF2) and the extent of IRR (27). In other words, certain tumours which are more difficult to cure by x-ray treatment may have more IRR. Note again that not all cells/organs need exhibit IRR. Application of these concepts for medical benefits includes:

· Development of a probe for IRR: to individualize treatment, we need to know which patient has a tumour with a large IRR, and that patients have sensitive normal tissue which must also be taken into account (which proteins/genes produce IRR? Many groups are using molecular approaches (refs in (8, 28)).
· Development of a sensitizing agent or treatment to prevent the development of IRR in the tumour such that the treatment follows the HRS portion (or "S" in Fig. 1) (7).
· Development of fractionation schemes for difficult cases: e.g. ultrafractionation for a tumour with large IRR should follow the HRS portion (again, S in Fig 1). A phase II study is in progress with encouraging results (low dose per fraction, skin metastases: Ph. Lambin, pers. comm.).
· A variation of this has been in use for some time - namely higher LET radiation such as neutrons or pions, which have since been found to exhibit less or no IRR (4, 29).
· Development of a class of protectors for normal tissue: turning on the protective response IRR in these tissues by priming with another type of insult. For example, could skin erythemia (30) presumably produced in the penumbra due to HRS, be avoided by prior priming with a chemical, or even UV?

A non-linear response to low doses of radiation has been seen in clinical response (human skin reaction) (30) as well as in vivo (kidney, skin, lung (31) and in vitro, above). A protective effect against human breast cancer by doses which prime IRR (10) (0.15-0.25 Gy) was noted (21). This analysis by Webster of data from (23) shows initially a similar shape to Fig 1.

Finally there may be similar examples in chemotherapy. The highly successful anti-cancer agent, cisplatin, exhibits similar structure to the above (cell survival vs. concentration, with a hypersensitivity to about 1 mM in RIF1 cells (32), or 2 mM in CHO cells (6)). This is being pursued elsewhere (A. Rainbow, pers. comm.; (16)). Chronic exposure of cells to cisplatin (0.01mM) increased resistance to higher concentrations, possibly eliminating the "structure" at low concentrations (6). If this occurs in patients and if this behaviour is exhibited by other agents, then, prior to adaptation, it might be feasible to administer lower concentrations to achieve the same toxicity. If, as above, one could somehow prime the normal tissue and not the tumour, an increased therapeutic index would result.

Clearly, a better understanding of AR's, IRR and hormesis involved in medical procedures (including surgery, drugs, hormones, radiation, heat, light for treatment and for diagnosic procedures) would be beneficial to human health.

4. How does the AR relate to the concept of hormesis? (and to IRR)?

If organisms respond by feedback mechanisms which may over-correct, etc., then the integration over time with such an array of parameters (timing, amplitude, duration, cell type, genetics) could produce virutally all of the reported shapes:

b-curve

U-shaped

IRR (fig 1)

as well as those predicted for "normal" dose responses.

In hormesis, the endpoint measured (e.g. cell survival, DNA repair, growth rate, etc.) is actually increased above control by the insult, whereas in IRR/HRS there is an initial decrease (Fig. 1). These two shapes have been seen using a common endpoint - namely colony size (Fig 1 of (3); Fig 3 of (33)). There is not necessarily a threshold for hormesis, while IRR appears to have a threshold and saturation. The range of the hormetic zone is 10x (3) while the IRR range is ~ 3x. Priming IRR (see "P" in Fig 1) is like the AR of Wolff - a small dose gives protection against a large dose (9).## In hormesis, the toxic agent stimulates in a more general sense. In addition, a result akin to hormesis (increased survival) was attributed to "recruitment" of cells which otherwise would have died; and the proposed "induced repair implies that the survival response is a discontinuous function of dose" (of low dose radiation) (17). Again, one goal of this edition could be to arrive at a unifying theory to cover these variations from the "norm". Control theory (3) is viewed as an alternate perspective on IRR and AR (in addition to hormesis). The experiments required to find evidence for or against will not be easy - seeing oscillations in the human environment, or even in mammalian cells, is challenging. Even in a controlled situation, such studies are very difficult (24). However, such a theory might suggest additional approaches and experiments to lend it support. Alternatively, existing models for IRR (variable a (4), AIDR (34), RMR (35)) might also be applied to AR or hormesis.

5. Should a knowledge of the AR affect current risk assessment methods for carcinogens?

It is clear that chemical carcinogens must be assessed at low concentrations, just as effects of radiation must be examined at low doses. The technology may be limiting but we must continue to push on these limits, rather than continuing to extrapolate from the easier experiments at high doses. A chemical may have one activity at concentration c and quite a different effect at 100 c, due to saturation of enzymes, genes turned on or off, inhibition of transport etc. The editor of this Newsletter has clearly devoted considerable efforts in these areas (36) but we do not yet know enough about AR to modify the entire set of guidelines.

In the meantime, we are also learning about genetic predispositions, and these must be considered in risk situations. One example was given recently: moving those miners who were reacting to the chemical insults deep in the mine to higher locations with lower levels of such agents. Another example might be screening astronauts for genetic predisposition, i.e. radiation sensitivity. However, such ethical issues will require considerable input from philosophers.

Acknowledgments:
The author is grateful to NCI of Canada for financial support of studies on low doses of radiation, and to Jack Beveridge for discussions on the Stebbing papers on control theory, and to colleagues and collaborators in this field.

FOOTNOTES

* Dr. Wolff received the first Leonard Sagan Award, 1998, from the BELLE society for his work in this area (W. Morgan, BELLE Newsletter 7 #1 page 36-37, 1998) as well as an earlier Failla award from the Radiation Research Society (2).

# Stebbing definition- "stimulatory effect caused by low levels of toxic agents" (3)

** also suggested in the Nova Scotia population, after fluoroscopy, where breast cancer was 3-4 times higher than total Canadian patients (21, 23). The incidence of AT heterozygots is proposed to be high in NS.

## Note however that differences in details between AR and IRR do exist (8), but this should not be surprising. IRR may also be similar to hormesis, as small doses of toxic agent improve survival, but IRR may require an accumulated level of damage, and usually does not give survival >1.

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