ARTICLE IN PRESS

Journal of Theoretical Biology 244 (2007) 489–499

www.elsevier.com/locate/yjtbi

The notion of hormesis and the dose–response theory:

A unified approach
M.A. Murado, J.A. Vázquez
Grupo de Reciclado y Valorización de Materiales Residuales, Instituto de Investigacións Mariñas (CSIC), r/Eduardo Cabello, 6, 36208 Vigo, Galicia, Spain
Received 17 March 2006; received in revised form 13 July 2006; accepted 6 September 2006
Available online 12 September 2006

Abstract

According to an opinion which is vigorous and insistently defended for approximately one decade, hormesis (the response of a
biological entity to an effector, with stimulatory results at low doses and inhibitory results at high doses) radically puts into question the
classic theory of dose–response (DR) relationships and demands a profound revision of environmental protection policies. Herein we
show that DR theory, with the modifications which we propose, allows the modelling of various kinds of biphasic responses which are
phenomenologically similar to hormetic ones and of well-defined origin, as well as responses which have been treated as genuinely
hormetic. Our descriptive approach may also represent a useful resource for experimental design, directed towards identifying some of
the potentially heterogeneous mechanisms which underlie the hormetic phenomenon. Finally, it also allows to discuss some factors which
prevent the use of the notion of hormesis—perhaps useful in a clinical context, under strictly controlled conditions—to make decisions
on environmental protection measures.
r 2006 Elsevier Ltd. All rights reserved.

Keywords: Dose–response; Generative model; Hormesis

1. Introduction lower than that accepted as maximum dose without effect

The classic approach to the relationships between the
dose of an effector and the biological response which it
causes (dose–response, or DR relationships) has been
attacked over the last years by numerous studies at whose
core is the phenomenon of hormesis (Calabrese and
Baldwin, 2003a, b; Calabrese, 2005; Ellman and Sunstein,
2004). Hormetic response is defined as that in which an
effector is beneficial at low doses and harmful at high doses
(e.g. antitumoral and tumorigenous), or, more generally,
that with a biphasic profile which changes not only in
magnitude, but also in sign, on varying the dose of the
effector. The aforementioned criticism maintains that
hormesis is much more common than current toxicology
admits (Calabrese and Baldwin, 2001; Calabrese and
Baldwin, 2003a, b), and in its favour adduces the frequency
with which various systems produce responses at doses
Corresponding author. Tel.: +34 986 231930; fax: +34 986 292762.
E-mail address: recicla@iim.csic.es (M.A. Murado).
with regard to the control (NOAEL: no observed adverse
effect level). Whereas at doses lower than the NOAEL, the
DR theory predicts only random variability with a null
average, examination of numerous experimental results
(Calabrese and Blain, 2005) seems to reveal that in a large
proportion of cases, responses of a type opposite to that
expected arise, whose statistical significance and distribu-
tion along the dose axis prevent them from being
considered as aleatory (Fig. 1).
The upholders of the hormetic response point out that its
identification is hindered by its habitual manifestation at
very low doses and with much less intensity than the
opposite response, as well as by an experimental practice
which often tests too few doses in too limited intervals. At
the same time they maintain that the main obstacle for
recognition of the phenomenon is the dogmatism of the
classic theory (Calabrese and Baldwin, 2000; Calabrese,
2004), together with the incorrect assimilation of hormesis
into the questionable homeopathic doctrine (which even
mentions dilution factors higher than Avogadro’s number,

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doi:10.1016/j.jtbi.2006.09.002
 ARTICLE IN PRESS
490 M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499
NOAEL
(A)
(B)
(C)
Fig. 1. Classic (A) and hormetic (B, C) dose–response relationships. The
criterium for admitting hormesis is based on the existence, at doses lower
than NOAEL, of responses statistically not null and of opposite sign to
those detected at doses higher than NOAEL (the shady band indicates the
confidence interval considered). But it would be also necessary to demand
that these responses can be formally described (dotted lines) by means of a
DR model appropriately generalized.
thus making statistically impossible the presence of the
effector in the homeopathic preparations). Finally, they
add that current environmental protection policies against
pollution, based on the classic theory, are unnecessarily
expensive and should be revised in the light of the hormetic
perspective (Calabrese, 2004; Teeguarden et al., 2000).
The criticism, in principle supported by wide data
reviews (Calabrese and Blain, 2005), is correct in pointing
out the inhibitory effects which a particular conceptual
paradigm may exert in a scientific field. However, as has
also been underlined (Kuhn, 1970), a series of anomalies is
not sufficient to invalidate a theory while the isolated cases
are not framed in a more general paradigm. Among the
efforts to make the concept of hormesis scientifically
acceptable, the search for explanatory mechanisms, for
unified phenomenological models, or for criteria for
discerning between different casuistries is noticeably
lacking. And the generalizations drawn regarding environ-
mental policies appear to be risky at least. Below we shall
try to discuss the different facets of the problem which the
notion of hormesis arouses.
2. The biphasic response and its formal description
The fact that a variable may produce, in different regions
of its domain, opposite effects on another, is a common
phenomenon in many spheres of reality. The absorption of
water is essential for life, but lethal above a certain level.
An increase in the atmospheric concentration of CO2 is
favorable as it activates photosynthesis, but it threatens us
through the greenhouse effect. In a more basic field, a
reduction in distance increases interatomic attraction, up to
a point where it causes repulsion. In these cases, and in
many others, the apparent paradox is due only to the fact
that a variable either intervenes in several phenomena at
the same time (those involved in the limits of cellular
stability, in the case of the water), or acts as the vehicle of
another (temperature, in the case of CO2), or interacts with
different entities (atomic cortex and nucleus, in the case of
the forces).
Physicists have no qualms about adding algebraically the
mathematical models which describe the phenomena
involved in this type of biphasic responses (as they do for
interatomic forces, where they add a hyperbolic equation
and another negative exponential). Analogously, when in a
biological context, contrasting phenomena in response to
the variation of a variable are detected, one might expect
the formal description to demand the addition of two
models. What kind of models?
The classic DR theory rests on equations which generate
sigmoidal profiles, and whose essential elements, under
various mathematical forms, are three-fold: the slope of the
response, its asymptotic maximum and the dose (RD50)
corresponding to the semi-maximum response. We will
enter into more detail below, but for the moment we will
only point out that the sigmoidal character is not a dogma,
but a necessary consequence of the DR phenomenon.
Indeed, if a DR model describes the response of a
population of biological entities to an effector, and said
response is quantified as R ¼ E=Y (E being the elements of
a population Y which respond to a given dose), in the
event—a common event—that the sensitivity to the effector
is distributed among the population according to an
unimodal density function, the DR model, which is nothing
but the corresponding accumulative function, will necessa-
rily be sigmoidal. In a previous work (Murado et al., 2002),
we have built, starting from very elementary hypothesis
(see Box 1), an algorithm which simulates population
responses to doses of an effector, showing that, even if
there are various factors which affect the specific forms of
the resulting relationships in characteristic ways, the
sigmoidal profile is always the basic module of the
phenomenon.
When two sigmoids are added or subtracted, the result
depends on the relationships between the parameters which
represent the aforementioned elements, affording the
variety shown in Fig. 2 (the use of logistic equations for
this is not essential; other sigmoidal equations lead to the
same situations). Some of the profiles thus obtained differ
 ARTICLE IN PRESS
M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499 491

Box 1
Four simple hypothesis that allow to simulate the response of a population of any biological entities to the
doses of an effector (Murado et al., 2002)

H1. Each biological entity has n potential receptors of the effector, and each receptor may adopt two
different states (active or sensible and inactive or immune), a being the number of active receptors.
H2. The dose D is defined as the number of effector units (molecules) present in each cell, accepting that
every molecule is capable of bonding with one receptor.
H3. Restricting the effect E to two modalities: death (E ¼ 0) and survival (E ¼ 1), and assuming that the
lethal effect occurs when at least p receptors are occupied, the following rule can be established:
(
If Dop; E ¼ 1;
If aop; E ¼ 1 8D: If aXp
If DXp; E ¼ 0:
Thus, when of a total number of biological entities Y, M dies at a given dose, the population
response can be formulated as R ¼ M/Y. It must be noted that direct comparison between D and p is
not pertinent. With aopoD, we will find E ¼ 1 at any dose, because aop, even though D4p. This
allows for the possibility of the existence of elements that are immune to the effector (aop, although
n4p), which is an essential condition of the DR models.
H4. Finally, it is possible to consider two kinds of additional factors, capable of affecting the response, as
they induce changes in either the receptor or the effector or in both. When they affect the receptor, we
can talk of excitors and inhibitors (h), depending on whether they raise or lower the value of a. When
they affect the effector, we can talk of enhancers and antagonists (f), depending on whether the
effective dose De they produce is higher or lower than the real one.

Under such hypothesis, and since any DR experiment implies populational values, n, a and p must be
considered as random numbers—in principle discrete—that are due to the variability of biological entities.
In addition, h and f must be assumed to be random—continuous in principle—that are due to the
experimental (or observational) conditions and the states of the effector.

little from the classic DR and may indeed be reasonably
fitted to a logistic equation, in particular if they are
associated to a slight experimental variability. Others go
further and suggest—if it is accepted that they are not only
mathematical artefacts—some interference in the classic
model. Lastly, others present the typical characteristics of
the hormetic response, there being cases where, at least in
theory, one might even speak of a triphasic response.
3. Two biphasic responses
Although a mathematical description contributes to the
understanding of a phenomenon, it is obvious that it does
not explain the same. And if the biphasic response may be
described as the linear combination of two sigmoids, there
still remains the question of its causes. To discuss this, we
shall commence by examining two particular examples of
this type of response.
Example 1. Many lactic acid bacteria (LAB) are involved
in probiotic interactions, i.e. a set of physiological effects
favorable for a host biological entity, linked to the
microbial balances of its intestinal tract (Fuller, 1990).
Probiotic effects have been related with the stimulation of
the natural defences through mechanisms as the competi-
tion with pathogenic microorganisms for limiting nutrients
or adherence points to the mucous, the activation of the
immunitary response or the production of inhibitory
substances. Among these last ones, it is necessary to point
out bacteriocins (recently revised by Riley and Wertz,
2002), antibiotic peptides with more favorable properties
than classic antibiotics for certain uses such as, for
example, the preparation of diets for animal feeding. In
fact, diets supplemented with LAB or bacteriocins are at
the present time a resource to reduce the high larval
mortality in fish farming, in many cases attributed to the
colonization of the intestine by unwanted microbiota
(Villamil et al., 2003).
In this context, a method for verifying the potential
interest of a LAB consists of measuring the inhibitory
effects of its cell-free culture medium on a pathogenic
microorganism which is habitual in the target species. A
bioassay of this type (Vázquez et al., 2005), with
Lactococcus lactis against the pathogen Vibrio alginolyticus
isolated from turbot, gave a clear biphasic response,
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492 M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499

m1 = m2 m1 < m2 m1 > m2

(A) (B) (C)

1+2

1 = 2 1-2

(D) (E) (F)

1 < 2

(G) (H) (I)

1 > 2

Fig. 2. DR profiles (continuous lines) obtained by lineal combination of two logistical equations (dotted lines 1 and 2). The asymptote of the first equation
is always higher than that of the second one (K14K2). The relationships between slopes (mi) and abscissas for semi-maximum response (RD50 ¼ mi ) are
indicated on the figure. Notice that substractive profiles C, G and I obey the conditions of the hormetic response, and that the case F could be defined as a
triphasic response. In connection with the remaining combinations, see text.

1.0
1
0.8
0.8
0.6

0.4
response

0.6
response

0.2
0.4
0.0

-0.2 0.2

-0.4
0
-0.6 0 0.5 0 0.5 1
0.0 0.1 0.2 0.3 0.4 bacteriocin (AU)
dose
Fig. 4. Inhibition of Carnobacterium piscicola by cell free extracts of
Fig. 3. Response of Vibrio alginolyticus to cell free extracts from Lactococus lactis (left) and Lactobacillus casei (right). Experimental results
Lactococcus lactis. Doses represent dilutions of the post-incubated culture (points; confidence intervals for a ¼ 0:05; n ¼ 4) fitted to additive logistic
medium of L. lactis that were combined with an equal volume of a models (continuous lines). AU: bacteriocin arbitrary units.
V. alginolyticus culture, with an initial absorbance (700 nm) of 0.200
(exposition time ¼ 6 h). Positive response corresponds to the (expected)
inhibitory effect; negative response represents stimulatory effect. Experi- adjustable to a difference of sigmoidal functions (Fig. 3).
mental results (points; confidence intervals for a ¼ 0:05; n ¼ 4) fitted to a But a medium concentrated by dialysis, retaining the
substractive logistic model (continuous line). peptide, provoked a drop in response instead of the

M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499
expected increase. And the suspicion that the effect was, in
fact, due to low residual levels of other metabolites of
L. lactis (lactic and acetic acids) was confirmed in
additional bioassays with pure acids (which reproduced
the inhibitory branch), with pure bacteriocin (with no
significant effect) and with an exhaustive dialysate of the
medium, which contained only non-dialysable peptides and
which reproduced the stimulatory branch. Therefore, the
biphasic response was due to the contrasting, independent
action of two types of effectors.
Example 2. In the preceding context, other media from
other LAB, when assayed against the pathogen Carnobac-
terium piscicola, repeatedly gave responses like those in
Fig. 4, adjustable to a sum of sigmoidal functions and
reproducible with pure bacteriocins (Murado et al., 2002).
Therefore, in this case, no more than one effector could be
assumed. But this requirement is not necessary either.
The model in Box 1 allows us to show that if at least one
of the elements (n, p, a) of the response obeys an
asymmetric probability distribution, the DR profile is
biphasic for any distribution of the factor f. Now then, the
sensitivity to an effector—represented by p—often depends
on the non-additive interaction of many factors, a case in
which its distribution throughout a population will not be
normal, but approaching an asymmetric model, such as the
log-normal or Weibull’s equations—in fact, the physical
nature of n, p and a imposes a distribution with a domain
[0,N), and not (N,N). Sensitivities with bimodal or
multi-modal distributions would have similar conse-
quences.
On the other hand, f signifies the effective dose interval
De which corresponds to a nominal dose D, an interval
affected, naturally, by experimental error. But the equili-
brium between the states which an effector may adopt
when in interaction with the conditions of the medium,
such as temperature and pH, also have an influence. The
states of ionization in the side chains of peptidic molecules,
such as bacteriocins, could be an example of this, where the
affinity for the receptor would thus be a random variable,
in a way assimilable to an internal energy which, like the
average rate in a system of particles, may be described by
0.8
response
0.6
0.4
0.2
25°C
0
0 5 10 15
Fig. 5. Effect of temperature on the biphasic profile of the inhibition of Carnobacterium piscicola by pure nisine. Experimental results (points; confidence
intervals for a ¼ 0:05; n ¼ 4) fitted to additive logistic models (continuous lines).
ARTICLE IN PRESS
493
means of the Maxwell distribution. In such a case, it could
be expected that the temperature would affect not only the
response, but specifically its biphasic profile, and this is
precisely what was proved by the bioassay with pure
bacteriocin (Murado et al., 2002) at different temperatures
(Fig. 5).
It should be added that the equilibrium between the
states of a single effector, even though very simple, might
explain phenomena of complex appearance within a DR
theory extended under the preceding terms. An organic
acid, such as lactic acid, is toxic to many biological entities
in non-ionic form, but not in ionized form (Gonc- alves
et al., 1997; Saha et al., 2006), the equilibrium between said
forms being a function of the pH. Thus, it is sufficient to
assume a receptor of the ionic form with different affinity
to the effector, and able to promote an activating
response—it may be sufficient to suppose a biological
entity capable of using lactate as a nutrient—for obtaining
a subtractive biphasic profile, as at a given pH the two
forms in equilibrium would be equivalent to two effectors.
In fact, it could even be assumed—speculatively—that this
is an usual phenomenon, but that it does not translate to
two-phase profiles more frequently because the probably
high averages of factors such as those represented by n, a
and p in Box 1 bring their distributions close to normal
(even being log-normal or Weibull’s), leading to very
smooth biphasic profiles which, in practice, are absorbed
by experimental error.
4. An example of hormesis
Many xenobiotic compounds induce in the receptor
enzymes with a relatively low substrate specificity which
may catalyse transformations in a second xenobiotic,
reducing its toxicity (or increasing it, as is the case with
organophosphorous insecticides), or may modify some
endogenous substrate, thus being able to alter some
metabolic equilibrium. This type of facts has been known
for some time and forms the basis of many studies, such as
a recent one (Kitano et al., 1998) on the role of
phenobarbital as promoter, in the rat, of hepatic carcino-
genesis initiated by diethylnitrosamine.
30°C 35°C
20 0 5 10 15 20 0 5 10 15 20 25
nisin (ppm)
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494 M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499

No more details are required to say that the effect of
phenobarbital, measured by means of two indicator
variables, was presented in the format of the histograms
in Fig. 6A, then considered by other authors (Teeguarden
et al., 2000) as an example of hormesis, and re-interpreted
according to Fig. 6B. In this last figure, the high doses were
disregarded in order to highlight the domain of the low
doses, a justifiable option, but which leads us to suppose,
for the response, a lower asymptotic level and at lower
doses than those deducible from Fig. 6A (a perception
accentuated by an error in transcription). In fact, if the
original data—values treated as categories—are treated as
values, Fig. 6C is obtained, where the possibility of
adjusting the results to subtractive DR models may be
seen, with some difficulties attributable to experimental
error.
Therefore, these results seem neither ‘‘to challenge DR
dogma’’ (Calabrese, 2005). From a formal point of view,
the response is treatable by means of the extension of the
classic theory proposed herein. From a material point of
view, it should be noted that the experimental design
involved two effectors (diethylnitrosamine and phenobar-
bital). Even when a single effector is used, if this acts as an
inducer of drug-metabolizing enzymes, the possibility of

14 1.4
number GST-P-positive foci area GST-P-positive foci
12 1.2
10 1.0

response
response

8 0.8
6 0.6
4 0.4
2 0.2
0 0
0
1
2
4
7.5
15
30
60
125
250
500

0
1
2
4
7.5
15
30
60
125
250
500
(A) phenobarbital (ppm) phenobarbital (ppm)

30
50
20
40
10
30
response (%)

response (%)

0
20
-10 10
-20 0
-30 -10
-40 -20
-50 -30
0 10 20 30 0 10 20 30
(B) phenobarbital (ppm) phenobarbital (ppm)

130 140
110 120
90 100
70 80
response (%)
response (%)

50 60
30 40
10 20
-10 0
-30 -20
-50 -40
0 50 100 150 200 250 300 0 50 100 150 200 250 300
(C) phenobarbital (ppm) phenobarbital (ppm)

Fig. 6. Three perspectives of two responses to fenobarbital (number and area of foci with positive reaction for glutathione S-transferase placental form) in
diethylnitrosamine-induced hepatic carcinogenesis in the rat. (A) the authors’ of the experiment presentation (Kitano et al., 1998). (B) line of union (spline)
of the responses (the dotted line represents a transcription error) in the low doses domain, according to an interpretation (Teeguarden et al., 2000) of the
results as a hormesis example. (C) Our fitting of the data to substractive logistic models (the points corresponding to 500 ppm, used in the calculations,
were suppressed of the graph because they do not modify the appreciation of the asymptotic level). Notice the different scales.
 ARTICLE IN PRESS
M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499
mechanisms able to generate secondary effector molecules,
or toxicologically relevant collateral responses should be
considered.
5. The doubtful novelty and the possible risk of the hormesis
notion
In the light of the precedent analyses, it is difficult to
avoid the idea that the hormesis notion simply constitutes a
particular (biphasic) case of the classic DR model. A case
whose empirically detectable manifestations suggests that
the phenomenon under study involves two effectors, two
receptors or, anyway, two mechanisms, each of which
strictly obeys a classic DR model. Other authors (Holladay
et al., 2005) have already pointed out that the stimulation
and inhibition arms of a biphasic curve may represent
mechanistically different events, like vitamin A deficiency
causes harmful visual effects, while excessive vitamin A can
damage the liver.
Thus, when a hormetic (i.e. biphasic) response is detected
and verified, it is necessary to suspect the presence in the
system, besides the effector under study, of another
relevant variable not considered in the experimental design.
To identify this variable it is important, of course, a good
knowledge of the system; but also the aid of formal
models, the parameters of which can translate quantita-
tively different aspects of the response with well-defined
biological meaning. These models are also essential tools
to organize the experimental work in such a way that
allows to decide if the results violate or not the DR theory.
Although the last example discussed in the precedent
section made possible this verification, it is not too frequent
in the bibliography about the hormesis. Anyway, no
practical decision should precede the clarification of the
mechanisms underlying the response or, at least, the
identification of the variables that give him its peculiar
form.
It is in this practical dimension where the concept of
hormesis can be more problematic, especially if it seeks to
justify the relaxation of the environmental protection
policies, at the present time based on the generally accepted
toxicological theory. Indeed, the hormetic response can
only be considered as a phenomenon located beyond that
theory if it is supposed that it is a function of a single
variable, a supposition that, already very doubtful under
reasonably controlled experimental conditions, becomes
untenable under environmental context. In this case it
would be not even be possible to enunciate criteria to
decide what it is considered as a beneficial response, since
the own qualification of beneficial is often ambiguous.
It is said, for example, that low doses of radiation may
be beneficial due to their possible immunostimulant effect
(Luckey, 1982), but this effect would be undesirable should
it stimulate autoimmunity. Hepatic oxigenases induced by
many xenobiotics can have a detoxicant role; but—as it
was demonstrated by the environmental contamination by
organochlorinated pesticides—those enzymes also act on
495
endogenous metabolites, altering essential metabolic bal-
ances, as those that govern the setting in the birds. Finally,
hormetic responses often imply very asymmetric biphasic
curves, not only due to the low magnitudes of the doses
and responses involved—as the defiance thesis admits—but
in particular to the narrow interval of doses in which they
appear. Thus, although the establishment of a biphasic
response in a certain natural subsystem would be doubtless
of interest, it could not really serve as a secure basis for
changes in environmental protection measures, given the
great variety of physical, chemical and biological mechan-
isms which redistribute the levels of xenobiotic molecules
among the different compartments of the environment.
6. Characteristics required for a DR model, and their
violations
It is still necessary to make a consideration with regard
to the modelling of DR relationships, and not only in order
to specify the adjustments which we have applied to the
preceding examples. Defining a DR model as the descrip-
tion of a population response to an effector is equivalent to
imposing him three conditions: (1) sigmoidal profile,
already discussed, (2) absence of intercept, to translate
the absence of effect at null dose, (3) a not necessarily
unitary asymptote (the totality of the population), to
translate the possible existence in the population of
resistant or immune elements. Among the various possible
mathematical solutions to this problem, for both practical
and theoretical reasons, those which involve few para-
meters with well-defined physical (biological) significance
are preferable. On detailed examination (Murado et al.,
2002) of several common DR models, the three which
appear in Table 1 proved particularly useful, other options
being either equivalent under their formal differences, or
promoters of scarcely relevant fitting improvements, with
the drawback of adding parameters with no clear biological
significance.
However, none of the expressions in Table 1 fulfills all
the conditions required for a DR model. In the Verhulst
(logistic) and Gompertz equations, the intercept must be
subtracted, and the Weibull equation requires the insertion
of a factor which represents an asymptote other than 1. But
once thus modified, in the first two (not in the third) the
significance of some parameters is only apparent. K no
longer translates the maximum response, nor do the
expressions of the center column of Table 1 define the
dose for the semi-maximum response (RD50 ¼ m), a key
datum for DR analysis. Therefore, the real maximum
response (Kr) must be determined by calculating the limit
of each function when D-N, and the real RD50 (mr)
finding the value of D when R ¼ K r =2. Furthermore, it is
recommended to reparameterize the functions in order to
make explicit m, using the expressions of m (Table 1, centre
column) to isolate another parameter, and insert the result
into the corresponding equation. Table 2 shows the models
thus modified, the first of which (logistic) was the one
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496 M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499

Table 1
Three usual formulations of DR relationships

Equation RD50 ( ¼ m) Asymptote (Rmax) and intercept (R0)

K
Verhulst R ¼ 1þexpðcmDÞ ; c ¼ ln½ðK=R0 Þ  1 m ¼ mc K
Rmax ¼ K; R0 ¼ 1þexpðcÞ
Gompertz R ¼ K exp½ expðb  cDÞ blnðln 2Þ Rmax ¼ K; R0 ¼ K exp½ expðbÞ
m¼ c
Weibull R ¼ 1  exp½ðD=yÞa  m ¼ yðln 2Þ 1=a Rmax ¼ 1; R0 ¼ 0

The parameter m in the Verhulst’s equation translates the maximum specific response (increment of R per unit of R and per unit of D, with dimensions
D1). K translates in all the cases the maximum response. The rest of the parameters only has formal meaning, the relationship of which with RD50 is
specified in the central column.
Note: Substituting (cmD) for (mDc) in the Verhulst’s (logistic) equation, we obtain the Fermi’s equation, which represents the (descending) complement
of the logistical response. If a ¼ 1, the Weibull’s equation becomes the von Bertalanffy’s equation.

Table 2
Equations of Table 1, modified so that they meet the basic conditions of a DR model (sigmoidal with R0 ¼ 0 and Krp1) and reparametrised to make
explicit the apparent RD50, or m

Equation Maximum response (Kr) and RD50 (mr)

n o h i
1 1 expðmmÞ
R¼K 1þexp½mðmDÞ  1þexpðmmÞ K r ¼ K 1þexpðmmÞ
n o
mr ¼ ln½2þexpðmmÞ
m
a
R ¼ Kfexp½ expðh þ cðm  DÞÞ  exp½ expðh þ mcÞg K r ¼ Kf1  exp½ expðh þ mcÞg a
n h  io
a
mr ¼ 1c h þ mc  ln ln 1þexp½ expðhþmcÞ
2
   a  b
R ¼ K 1  exp h D
m
Kr ¼ K
mr ¼ m
a
h ¼ ln(ln 2).
b
h ¼ ln 2.

applied to all the cases discussed herein, and the final
appendix details the corresponding mathematical treat-
ments.
However, all too frequently, these models—or others of
similar implications—are used in their original formats
(Table 1), even linearized when possible. In fact, the logistic
equation is the basis of probitic transformation (Bliss,
1937; Finney, 1952), a classic resource for the linearization
of a DR curve. Although sometimes useful, said lineariza-
tion is not satisfactory if Ko1 (populations with a resistant
fraction), as two cases with a different K generate
indistinguishable straight lines. But even with non-linear
fitting methods, a function with an intercept R06¼0 poses
problems with data affected by experimental error, as the
solution tends to imply an unacceptably high R0 and biases
in the other parameters. The problem becomes reduced by
inserting restrictions which limit R0 to very low values with
regard to K, but this makes the slopes highly sensitive to
experimental error and increases the lack of fitting at low
doses. Perhaps these difficulties (which can be easily
overcome) often discourage the formalization of DR
results and lead to simplistic extrapolations like the ones
sometimes pointed out by those who maintain that
hormesis represents an anomaly which exceeds the
possibilities of current quantitative toxicology.
Naturally, the formal tools can say nothing conclusive
about the facts. However, the formalism of the DR theory
is so directly linked to the basic facts of the phenomenon
that it is especially useful for contrasting hypotheses,
detecting anomalies and guiding experimentation in search
of mechanisms to explain each particular case.
7. Conclusions
For a little more than a decade, it has been emphasized
that hormesis demands a profound revision of the DR
theory, and important changes in protection measures
against environmental pollution. Without denying the
existence of biphasic responses, the arguments presented
herein suggest a different point of view, according to
which:
1. Biphasic responses, in a wide sense, may be due to
mechanisms of a very diverse nature. But this does not
invalidate the DR theory, which—with the additions
that we propose—is still useful for describing said
responses in a way that allows us to delimit them, to
calculate the toxicological parameters which are relevant
for practical purposes, and to direct experimentation in
order to reduce the phenomenon to known facts, or to
look for explanations in terms of new mechanisms.
2. The hormesis, when is not obviously reducible to a
combination of effectors, generally occurs not only at
low doses, but also within a very narrow interval in
 ARTICLE IN PRESS
M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499 497

relation to the domain which is relevant for the This formal feature lacks toxicological meaning (the
response. If we add to this the heterogeneity of the response should be null at null dose) and it can generate
mechanisms potentially involved, their interaction with biases in the values of the rest of the parameters calculated
the populational distributions of the sensitivity factors, by any fitting method.
the differences in sensitivity of different species and the Applying the same reasonings to the other two models,
physical, chemical and biological factors which redis- we obtain:
tribute the wastes from human activity into different A2: Gompertz equation:
compartments of the environment, it becomes difficult R ¼ K exp½ expðb  cDÞ.
to accept that the notion of hormesis may justify the
relaxation of the restrictions on pollutant emissions. A2.1: Maximum response (Rmax):
Rmax ¼ lim K exp½ expðb  c1Þ,
It is possible, certainly, that some hormetic responses D!1

may have a clinical application under strictly controlled ¼ K exp½ expð1Þ ¼ K.

conditions. But environmental conditions include not only A2.2. Dose for semi-maximum response (RD50 or m):
too many variables with ill-known effects, but also many
variables with implications of well-known risks. The Rmax K
¼ ¼ K exp½ expðb  cmÞ;
bioconcentration mechanisms of chlorinated insecticides 2 2
and polychlorinated biphenyls, terphenyls and naphtha- b  lnðln 2Þ
so that m ¼ .
lenes, the production of dioxins from chlorophenoxyacetic c
herbicides and other materials, or the diffusion of Inconvenience of non-null intercept:
resistance to antibiotics among bacterial populations are R0 ¼ lim K exp½expðb  c0Þ ¼ K exp½ expðbÞ.
only three examples of the fact that even a DR model D!0
which has been thoroughly verified in the laboratory is of A3: Weibull equation:
little predictive value when the receiving entity is the system
we call our environment. R ¼ 1  exp½ðD=yÞa .
A3.1: Maximum response (Rmax):
Acknowledgment Rmax ¼ lim 1  exp½ð1Þa  ¼ 1.
D!1

Dr. J.A. Vázquez had a postdoctoral contract (CSIC- A3.2. Dose for semi-maximum response (RD50 or m):
I3P-PC 2003) financed by the European Social Fund.
Rmax 1
¼ ¼ 1  exp½ðm=yÞa ; so that m ¼ yðln 2Þ1=a .
2 2
Appendix A
In this case the intercept is null:
Equations shown in Table 1 are of common use in the R0 ¼ lim 1  exp½ð0Þa  ¼ 0,
D!0
treatment of DR experiments and, in principle, their
parameters translate—directly or through simple relation- but another inconvenience arises: the maximum response is
ships—toxicologically relevant concepts. This way: always 1, what demands a modification to translate the
A1: Verhulst (logistic) equation is possible existence of immune elements in the target
population.
K
R¼ , When we insert in these models the necessary modifica-
1 þ expðc  mDÞ tions to eliminate their concrete inconveniences, we obtain
a formulation in which the following values are essential in the equations shown in Table 2. But these equations—
DR relationships: which now fulfill the three basic conditions of a DR
A1.1. Maximum response, or limit of the function when model—should be treated in the same way than the original
D-N: ones, to determine the new expressions that define the
K maximum response (Kr) and the dose for the semi-
Rmax ¼ lim ¼ K. maximum response (mr).
D!1 1 þ e1
B1: Modified logistic (with R0 ¼ 0 and Krp1):
A1.2. Dose for semi-maximum response (RD50 or m), or

value of D when R ¼ Rmax =2: 1 1
R¼K  .
1 þ exp½mðm  DÞ 1 þ expðmmÞ
Rmax K K c
¼ ¼ cmm
; so that m ¼ . B1.1: Maximum response (Rmax or Kr):
2 2 1þe m


However, the logistic equation has the inconvenience of 1 1
Rmax ¼ K r ¼ lim K  ,
its non-null intercept. Indeed, D!1 1 þ exp½mðm  1Þ 1 þ expðmmÞ
  mm
K K 1 e
R0 ¼ lim ¼ . ¼K 1 ¼K .
D!0 1 þ ec0D 1 þ ec 1 þ expðmmÞ 1 þ emm
 ARTICLE IN PRESS
498 M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499

B1.2. Dose for semi-maximum response (RD50 or mr): B3.1: Maximum response (Rmax or K r ¼ K):

 Rmax ¼ K r ¼ lim K 1  exp½hð1Þa  ¼ K.
Rmax emm D!1
¼K ,
2 2ð1 þ emm Þ B3.2: Dose for semi-maximum response (RD50 or


1 1 mr ¼ m):
¼K  ,
1 þ exp½mðm  mr Þ 1 þ expðmmÞ Rmax K 
emm emm  emðmmr Þ ¼ ¼ K 1  exp½hðmr =mÞa  ,
) ¼ , 2 2
2ð1 þ e Þ ð1 þ emðmmr Þ Þð1 þ emm Þ
mm 1
    ) ¼ exp½hðmr =mÞa  ) ln 2 ¼ hðmr =mÞa ,
) emm 1 þ emðmmr Þ ¼ 2 emm  emðmmr Þ , 2
 
1 ln 2 1=a
) mr ¼ lnðemm þ 2Þ. ) mr ¼ m ¼ m.
m h
Biphasic profiles are the direct result of the sum or
It should be noticed that the intercept is now null: difference of two modified models, not necessarily of the

 same type. These modified models are of particular utility
1 1 here, since the original ones always provide very poor
R0 ¼ lim K  ¼0
D!0 1 þ exp½mðm  0Þ 1 þ expðmmÞ fittings. Not all the toxicologically interesting values can be
obtained in all the phases through analytic expressions.
B2: Modified Gompertz (with R0 ¼ 0 and Krp1): However, once a set of experimental data is treated by
 means of any non-linear fitting method (e.g. quasi-Newton)
R ¼ K exp½ expðh þ cðm  DÞÞ  exp½ expðh þ mcÞ . and, this way, well-known the parameters of the model,
any value of interest can be calculated by numeric iteration
B2.1: Maximum response (Rmax or Kr): (Vázquez et al., 2005), or through search methods (again
quasi-Newton) based on the expected values of the

Rmax ¼ K r ¼ lim K exp½ expðh þ cðm  1ÞÞ response.
D!1

 exp½ expðh þ mcÞ ¼ K 1  exp½ expðh þ mcÞ . References

B2.2: Dose for semi-maximum response (RD50 or mr): Bliss, C.I., 1937. The calculation of the time–mortality curve. Ann. Appl.
 Biol. 24, 815–824.
Rmax K 1  exp½ expðh þ mcÞ Calabrese, E.J., 2004. Hormesis: a revolution in toxicology, risk
¼ , assessment and medicine. EMBO Rep. 5 (Special issue), 537–540.
2 2
 Calabrese, E.J., 2005. Challenging dose–response dogma. Scientist 14,
¼ K exp½ expðh þ cðm  mr ÞÞ  exp½ expðh þ mcÞ , 22–23.
) 1  exp½ expðh þ mcÞ þ 2 exp½ expðh þ mcÞ, Calabrese, E.J., Baldwin, L.A., 2000. The marginalization of hormesis.
Hum. Exp. Toxicol. 19, 32–40.
¼ 2 exp½ expðh þ cðm  mr ÞÞ, Calabrese, E.J., Baldwin, L.A., 2001. The frequency of U-shaped dose


1 þ exp½ expðh þ mcÞ responses in the toxicological literature. Toxicol. Sci. 62, 330–338.
)  expðh þ cðm  mr ÞÞ ¼ ln , Calabrese, E.J., Baldwin, L.A., 2003a. The hormetic dose–response model
2

  
 is more common than the threshold model in toxicology. Toxicol. Sci.
1 2
) mr ¼ h þ cm  ln ln
c 1 þ exp½ expðh þ mcÞ
The intercept is now null:
 overview. Toxicol. Appl. Pharmacol. 202, 289–301.
R0 ¼ lim K exp½ expðh þ cðm  0ÞÞ
D!0
 exp½ expðh þ mcÞ ¼ 0.
B3: Modified Weibull
In this case, the original equation—the intercept of
which is already null—only requires the insertion of a
factor (K) which represents an asymptote other than 1.
Values of the maximum response and dose for the semi-
maximum response do not vary as a consequence of this
modification:
71, 246–250.
.
Calabrese, E.J., Baldwin, L.A., 2003b. Toxicology rethinks its central
belief. Nature 421, 691–692.
Calabrese, E.J., Blain, R., 2005. The occurrence of hormetic dose
responses in the toxicological literature, the hormetic database: an
Ellman, L.M., Sunstein, C.R., 2004. Hormesis, the precautionary
principle, and legal regulation. Hum. Exp. Toxicol. 23, 601–611.
Finney, D.J., 1952. Probit Analysis. Cambridge University Press, Cam-
bridge.
Fuller, R., 1990. Probiotics in agriculture. Agbiotech 2 (2), 217–220.
Gonc- alves, L.M.D., Ramos, A., Almeida, J.S., Xavier, A.M.R.B.,
Larrondo, M.J.T., 1997. Elucidation of the mechanism of lactic acid
growth inhibition and production in batch cultures of Lactobacillus
rhamnosus. Appl. Microbiol. Biotechnol. 48, 346–350.
Holladay, S.D., Ehrich, M., Gogal Jr., R.M., 2005. Commentary on
hormetic dose–response relationships in immunology: occurrence,
quantitative features of the dose–response, mechanistic foundations,
and clinical implications. Crit. Rev. Toxicol. 35 (2/3), 299–302.
  a
 Kitano, M., Ichihara, T., Matsuda, T., Wanibuchi, H., Tamano, S.,
D
R ¼ K 1  exp h . Hagiwara, A., Imaoka, S., Funae, Y., Shirai, T., Fukushima, S., 1998.
m Presence of a treshold for promoting effects of phenobarbital on
 ARTICLE IN PRESS
M.A. Murado, J.A. Vázquez / Journal of Theoretical Biology 244 (2007) 489–499
diethylnitrosamine-induced hepatic foci in the rat. Carcinogenesis 19
(8), 1475–1480.
Kuhn, T.S., 1970. The Structure of Scientific Revolutions, second ed.
Chicago University Press, Chicago.
Luckey, T.D., 1982. Physiological benefits from low levels of ionizing
radiation. Health Phys. 43, 771–789.
Murado, M.A., González, MaP., Vázquez, J.A., 2002. Dose–response
relationships: an overview, %a generative model and its application to
the verification of descriptive models. Enzyme Microb. Technol. 31,
439–455.
Riley, M.A., Wertz, J.E., 2002. Bacteriocins: evolution, ecology and
application. Annu. Rev. Microbiol. 60, 117–137.
499
Saha, N.C., Bhunia, F., Kaviraj, A., 2006. Comparative toxicity of three
organic acids to freshwater organisms and their impact on aquatic
ecosystems. Hum. Ecol. Risk Assess. 12, 192–202.
Teeguarden, J.G., Dragan, Y., Pitot, H.C., 2000. Hazard assessment of
chemical carcinogens: the impact of hormesis. J. Appl. Toxicol. 20,
113–120.
Vázquez, J.A., González, MaP., Murado, M.A., 2005. Effects of lactic
% microbiota from fish. Aquaculture 245,
acid bacteria on pathogenic
149–161.
Villamil, L., Figueras, A., Novoa, B., 2003. Immunomodulatory effects of
nisin in turbot (Scophtalmus maximus L.). Fish Shellfish Immunol. 14,
157–169.