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Vol. 289, Issue 3, 1370-1375, June 1999
The College of Staten Island/City University of New York and College of Staten Island/Institute for Basic Research Center for Developmental Neuroscience, Staten Island, New York (B.K.); and Department of Psychology and Program in Neuroscience, University of Illinois at Urbana-Champaign, Champaign, Illinois (S.G.W., J.S.M.)
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Several variables have been reported to affect the expression of sex differences in the analgesic potency of morphine. Although the effect of genetic background on morphine analgesia has been well documented, the relevance of genotype to sex differences in morphine analgesia has rarely been considered. The present study investigated morphine dose-response relationships in male and female mice of 11 inbred mouse strains on the tail-withdrawal test after i.c.v. administration. Large differences in morphine analgesic potency were observed between strains, reflecting the important influence of genotype on this trait. We identified three strains (AKR/J, C57BL/6J, and SWR/J) in which males displayed approximately 3.5- to 7.0-fold greater sensitivities to the analgesic effects of morphine than did their female counterparts. In contrast, in the CBA/J strain, females were found to be approximately 5-fold more sensitive to morphine than were the males. In all other strains, morphine potency estimates between the sexes were not statistically different. These data support the importance of genotype, sex, and their interaction in the mediation of morphine analgesia and suggest that equivocal findings regarding opioid sex differences in the literature may be partially accounted for by the use of different subject populations. The fact that female mice of the AKR/J and CBA/J strains exhibit 35-fold different morphine analgesic potency and that males of these strains are equally sensitive should facilitate the mapping and identification of sex-specific genes of relevance to morphine analgesia.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The
analgesic effect of morphine in humans is subject to wide individual
differences (Lasagna and Beecher, 1954
), and it is becoming
increasingly appreciated that sex differences may contribute to this
variability (for reviews, see Berkley, 1997; Kest et al., 1998). Our
understanding of the contribution of sex differences in morphine
analgesia has been largely aided by research in rodent populations. For
example, the peak, total, and duration of morphine analgesia after
systemic administration have been shown to be greater in males than in
females in both rats (Baamonde et al., 1989; Islam et al., 1993; Cicero
et al., 1996) and mice (Kavaliers and Innes, 1987; Lipa and Kavaliers,
1990; Candido et al., 1992; Kavaliers and Innes, 1992a, b). A strong
case that sex differences in morphine analgesia may be mediated by
differential central nervous system mechanisms can be made based on the
observations that male rats display greater analgesic effect than
females after the administration of morphine into the cerebral
ventricles (Kepler et al., 1989) or the rostral ventromedial medulla
(Boyer et al., 1998).
However, the effect of sex on morphine analgesia remains equivocal
because several studies have failed to observe significant sex
differences (Kasson and George, 1984; Islam et al., 1993; Ali et al.,
1995). This lack of consistent findings may reflect methodological
differences between studies. Indeed, among the variables that have been
demonstrated to affect sex differences in opioid analgesia are dose
(Kepler et al., 1989; Bartok and Craft, 1997), nociceptive assay used
(Baamonde et al., 1989; Islam et al., 1993; Bartok and Craft, 1997),
postinjection testing latency (Bartok and Craft, 1997), time of testing
(i.e., circadian rhythmicity) (Kavaliers and Innes, 1987), and subject
age (Islam et al., 1993). Not usually considered is the well documented
fact that the analgesic potency of morphine can vary between
subpopulations of a single species (for a review, see Mogil et al.,
1996b). For example, large differences in morphine analgesia have been
reported between compared selectively bred, inbred, and recombinant
inbred mouse strains, reflecting a strong genetic component in the
mediation of morphine analgesic sensitivity. However, experiments
directly addressing the issue of whether the expression of sex
differences in morphine or opioid analgesia varies with genotype are
rare. One study reported that the magnitude of sex differences in
morphine analgesia (males > females) differed between
Sprague-Dawley and Wistar-Furth rats (Kasson and George, 1984). We
observed greater analgesia from the enkephalin-derived µ-opioid
receptor agonist [D-Ala2,N-MePhe4,Gly-ol5]-enkephalin
in male than in female mice selectively bred for high-, but not low-,
stress-induced analgesia (Kest et al., 1995). In an intriguing finding,
Rady and Fujimoto (1997) report that heroin analgesia is mediated
alternately by µ- and 
-opioid receptors, respectively, in ICR and
Swiss-Webster (SW) strains; this genetic difference displays
sex-influenced dominant inheritance, such that male (ICR × SW)F1 hybrids display the ICR phenotype, whereas female F1 hybrids display the SW phenotype.
Finally, endogenous opioid analgesic mechanisms, which are known to
represent the substrate on which morphine acts to produce pain
inhibition, are activated by forced cold-water swims in inbred male
DBA/2 mice but not in female mice of this same strain or in C57BL/6
mice of either sex (Mogil and Belknap, 1997). These studies all suggest that genotype (i.e., allelic differences at relevant genetic loci) may
contribute to sex differences in opioid analgesia and that the use of
different subject populations may be at least partially responsible for
the ambiguous state of the literature regarding these differences.
The aim of the present study was to begin a more comprehensive
examination of the putative interaction of genotype and sex in the
potency of morphine analgesia. Toward this end, morphine half-maximal
analgesic dose (AD50) estimates in male and
female mice of 11 inbred strains were compiled using the
tail-withdrawal test. To minimize the possible influence of sex
differences in morphine pharmacokinetic parameters (Candido et al.,
1992; Craft et al., 1996; but see Cicero et al., 1996, 1997), we used
the i.c.v. route of administration. The data demonstrate that the magnitude and direction of sex differences in morphine analgesia are
dependent on genotype.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Strains.
Naïve, adult mice of both sexes
(n = 8/sex/strain) of the following inbred strains were
obtained from The Jackson Laboratory (Bar Harbor, ME): 129/J, A/J,
AKR/J, BALB/cJ, C3H/HeJ, C57BL/6J, CBA/J, LP/J, SJL/J, and SWR/J. For
clarity, the J substrain identifier will be omitted. All mice were
shipped at 6 weeks of age (except BALB/c mice, which are only available
at 
4 weeks of age), housed four to a cage in a temperature-controlled
(22°C) environment, and maintained on a 12:12-h light/dark cycle
(lights on at 7:00 AM). Mice were allowed free access to food (Purina
chow) and water. Testing occurred at 7 to 8 weeks of age in all cases.
Morphine Administration.
Morphine sulfate was generously
supplied by the National Institute for Drug Abuse (Rockville, MD) and
was dissolved in 0.9% physiological saline. Injections were made into
the lateral cerebral ventricle according to the method of Haley and
McCormick (1957) with animals under oxygen/isoflurane inhalant
anesthesia. Briefly, a small midline incision was made in the scalp of
anesthetized mice, and the lambda was located. Drug then was injected
directly through the skull at a point 2 mm rostral and lateral to 
at a depth of 3 mm using a 10-µl Hamilton microsyringe with a
27-gauge needle. All injections were made in a volume of 5 µl. After
each injection, the incision was closed with a stainless steel wound clip.
Tail-Withdrawal Assay. Testing proceeded in two daily sessions near mid-photophase (10:00 AM to 12:00 noon; 2:00-4:00 PM) to reduce circadian effects on nociceptive and analgesic sensitivity. Because of availability restrictions and practical considerations, strains were tested in groups based on their arrival date (group 1: AKR, C3H/He, SJL, SWR; group 2: BALB/c, C57BL/6, CBA; group 3: 129, A, LP). Within each group, however, both strain and sex were completely counterbalanced, so one mouse per strain per sex was tested in each session.
After a 30-min habituation to the testing room, mice were assessed for baseline nociceptive sensitivity on the 49°C tail-withdrawal test. In this assay of acute, thermal nociception, the mouse is gently restrained, and the distal half of the tail is immersed in water maintained at 49.0 ± 0.2°C with an immersion circulator pump (Fisher Isotemp model 71). Latency to reflexive withdrawal of the tail was measured twice by an experimenter to the nearest 0.1 s, with each determination separated by 20 s. The two determinations were later averaged. The tail-withdrawal test was chosen because of its stability even after repeated exposures at this highly noxious water temperature. A cutoff latency of 15 s was used to prevent the possibility of tissue damage.Dose-Response Studies. Immediately after baseline latency assessment, morphine analgesic potency was determined, using cumulative dose-response curves to reduce the number of mice required. All subjects were injected with increasing doses of morphine (0.1, 0.5, 1.0, 2.0, 3.6, 6.5, and 11.7 µg) in succession. Tail-withdrawal latencies were retested 15 min after each injection, and subsequent doses of morphine were administered immediately after withdrawal latency assessment at each dose. This procedure was repeated until each mouse displayed a cutoff tail-withdrawal latency average of >15 s.
Data Analysis.
Analgesia at each dose was expressed as a
percentage of the maximum possible effect (%MPE) as calculated by the
formula: %MPE = [(postmorphine latency 
baseline
latency)/(cutoff latency baseline latency)] × 100. The use of
%MPE takes into account the cutoff latency and individual baseline
latencies, so these will not bias the quantification of analgesia.
). To evaluate
the genetic codetermination between thermal nociceptive sensitivity and
morphine analgesia, baseline withdrawal latencies and morphine AD50 strain means were correlated using
Pearson's r statistic. Variances of strain mean
AD50 values between sex were also compared using
a two-tailed F test. In all statistical tests, a criterion 
level of .05 was used.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Baseline Sensitivity on Tail-Withdrawal Test. Baseline tail-withdrawal latencies for all strains, between and across sex, are presented in Table 1. There was a highly significant main effect of strain (P < .001) and of sex (P < .001), and the interaction between strain and sex approached significance (P = .084). Indeed, pairwise comparisons of sex for each strain indicate that in three strains, AKR, C3H/He, and C57BL/6, males exhibited significantly higher baseline tail-withdrawal latencies than their female counterparts, reflecting lower nociceptive sensitivity. Thus, sex differences in baseline sensitivity on the tail-withdrawal test are genotype dependent. One-way ANOVAs performed on baseline withdrawal latencies for each sex indicate a significant effect of strain in both males (F10,75 = 5.90, P < .001) and females (F10,72 = 12.02, P < .001), although strain rank order sensitivity was not identical.
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Supraspinal Morphine Analgesia.
Cumulative dose-response
curves for all strains collapsed across sex are presented in Fig.
1, with corresponding
AD50 estimates (micrograms per mouse) and 95%
CIs presented in Table 1. We observed a wide range of potency estimates
between strains, confirming the relevance of genetic background on
morphine analgesic sensitivity. Figure 1 also shows cumulative
dose-response curves for all strains separately by sex. The larger
strain variability apparent for female mice is reflected in the
corresponding AD50 estimates for each sex in
Table 1. For example, although the largest difference in morphine
analgesic potency in males (approximately 14-fold) is observed between
the C3H/He and SJL strains, a 41-fold difference in morphine potency
distinguishes CBA and SWR females. Subjecting the ratio of the
variances to an F test confirmed that this variability between strain AD50 estimates in females was
significantly greater than that in males
(F10,10 = 3D 12.5, P < .001).
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0.61) and for males
(r = 0.40) and females (r = 0.68) alone, confirming in females the inverse relationship between initial
nociceptive sensitivity and morphine analgesia previously reported for
males (Mogil et al., 1996a; Elmer et al., 1997).
A separate experiment performed on AKR and CBA mice of both strains, in
which seven repeated i.c.v. injections of saline were made, revealed an
expected but modest (1-2 s) anesthesia/injection-related analgesia
(data not shown). Importantly, this analgesia did not increase in
magnitude with injection number and was statistically indistinguishable
in both sexes and strains. Thus, it is very unlikely that our
conclusions are confounded by sex or genotype differences in stress analgesia.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present findings clearly demonstrate that sex differences in morphine analgesia are genotype dependent. Although in the majority of the strains examined, there was no significant sex difference in the AD50 estimate for morphine analgesia, significantly greater analgesic potency was observed in male AKR, C57BL/6, and SWR mice relative to their female counterparts. The lack of sex differences in most strains may truly reflect the absence of such differences in these genotypes or our lack of statistical power to detect differences of modest magnitude. The present finding that female CBA mice were approximately 5.5-fold more sensitive to the analgesic effects of morphine than were males of this strain highlights the importance of genetic background on not only the magnitude but also the direction of sex differences.
That genotype should have an impact on sex differences in morphine
analgesia is consistent with previous reports that have considered sex
as a variable in genetic models of exogenous µ receptor-mediated
analgesia in rats (Kasson and George, 1984) and mice (Kest et al.,
1995). To our knowledge, sex differences in morphine analgesia have
without exception been examined in commercially available outbred
strains of rats and mice. Large phenotypic differences of relevance to
analgesia have been documented between different outbred strains and
even between populations of the same outbred strain maintained in
different breeding institutions (i.e., vendor effects) (Mogil et al.,
1996b). We suggest that variability in genetic background, perhaps
acting in concert with other variables (see the introductory
paragraphs), may have contributed to some of the conflicting findings
in the literature on sex differences in both basal nociceptive
sensitivity and opioid analgesia. To directly investigate this
possibility, the testing of common outbred strains would be required.
We instead chose to investigate only inbred mouse strains, the rodent
populations of greatest use for gene-mapping efforts that may shed
light on the responsible mechanisms (see below).
It is difficult to assess the accuracy of our
AD50 estimates because there are virtually no
instances in the literature in which the inbred mouse strains tested
here were administered morphine by the i.c.v. route. In the one
experiment, to our knowledge, in which more than one of these strains
were used, Frigeni et al. (1981) obtained morphine
AD50 estimates in male DBA/2 and C57BL/6 mice of
0.21 and 0.82 µg, respectively. These values cannot be easily
compared with the values obtained presently (0.36 and 2.02 µg,
respectively) because these investigators used a different nociceptive
assay, the 51.5°C hot-plate test. It is notable, however, that the
potency ratio between these strains from their and our studies are
comparable (4.0 and 5.6, respectively). Also engendering confidence in
the accuracy of the presently obtained data is the highly significant
correlation (r = 0.87, P = .005, eight
common strains) between baseline latencies of male mice in the present study and in a separate study conducted 1 year earlier in a different laboratory (Mogil et al., 1998).
Proposed Mechanisms Underlying Sex Differences in Morphine
Analgesia.
One obvious possibility is that morphine
bioavailability might differ between the sexes. Higher concentrations
of morphine in the brains of male rodents relative to females have been
observed after systemic injection in some (Candido et al., 1992; Craft et al., 1996) but not other (Cicero et al., 1996, 1997) studies. The
present demonstration of sex differences in morphine potency after
i.c.v. administration in mice, as previously reported in rats (Kepler
et al., 1989), argues against an exclusive role for pharmacokinetics in
accounting for the differential analgesic response of males and females
to morphine.
), which can affect morphine potency (Vaught
and Takemori, 1979), have been demonstrated in the rat (Watson et al.,
1986). With respect to opioid receptors, no consistent sex differences
in µ or opioid receptor populations are observed in rats (Kepler
et al., 1991). In mice, both increased levels in males (Mogil et al.,
1994) and no differences (Candido et al., 1992) in whole-brain opioid
binding between sexes have been reported. The disconnection between
analgesia and receptor density is well evidenced by the fact that male
mice exhibit a significantly greater morphine analgesic
supersensitivity than females after chronic treatment with the opioid
antagonist naltrexone despite the absence of sex differences in the
magnitude of opioid receptor up-regulation (Candido et al., 1992).
Attempts have also been made to relate sex differences in opioid
analgesia to gonadal hormone levels of male and female rodents. Although female rats are reported to display greater analgesic sensitivity to systemic morphine on the mornings of diestrus (Banarjee et al., 1983) and proestrus (Banarjee et al., 1983; Berglund and Simpkins, 1988), Kepler et al. (1989) reported a relative decrease in
sensitivity during met/diestrus. Studies with gonadectomized subjects
are even more conflicting. For example, although it appears that the
effect of adult ovariectomy on morphine analgesia may be nociceptive
modality specific (Baamonde et al., 1989; Ali et al., 1995), reductions
(Banarjee et al., 1983; Kepler et al., 1989), increases (Kasson and
George, 1984), and no alterations (Cicero et al., 1996) in morphine
analgesia have all been reported just for the tail-withdrawal test.
Like females, the analgesic potency of systemic morphine in castrated
adult rats (Chatterjee et al., 1982; Kasson and George, 1984; Ali et
al., 1995; Cicero et al., 1996) and mice (Candido et al., 1992; Ali et
al., 1995) is dependent on the nociceptive modality tested, and
contradictory findings are reported within the same assay
(tail-withdrawal test) for rats (Chatterjee et al., 1982; Kasson and
George, 1984; Cicero et al., 1996).
Finally, sex differences have been noted in other neurochemical systems
that have an impact opioid analgesia. For example, there is evidence
indicating that
N-methyl-D-aspartate-sensitive excitatory amino acid receptors mediate morphine analgesia but that
selective N-methyl-D-aspartate
receptor blockade significantly reduces morphine analgesia in males
only (Lipa and Kavaliers, 1990). Sex differences in the antiopioid
effects of the endogenous peptides Tyr-MIF-1 and neuropeptide FF on
morphine analgesia in mice have also been noted (Kavaliers and Innes,
1992a,b). However, the relevance of these findings remains highly
speculative. Overall, no clear consensus haso emerged regarding the
mechanisms underlying sex differences in morphine analgesia.
Application of Gene Mapping Strategies.
We believe that
further investigation of sex differences in opioid analgesia requires
the application of new strategies. We are attempting to delineate the
physiological mechanisms underlying differential opioid analgesic
sensitivity between the sexes by mapping the relevant genetic loci by
linkage analysis (Lander and Botstein, 1989). This approach, called
quantitative trait locus (QTL) mapping, can reveal chromosomal regions
(and, ultimately, genes) associated with continuously distributed
traits such as opioid sensitivity. QTL mapping has been successfully
used to identify loci associated with systemic morphine analgesic
magnitude on the hot-plate test using C57BL/6 and DBA/2 progenitors
(Belknap et al., 1995). However, several important issues remain
unexamined. First, these experiments examined morphine analgesia on the
hot-plate test, and we and others have determined that the genetic
mediation of this trait is at least partially specific to the
nociceptive assay used (Mogil et al., 1996a; Elmer et al., 1997).
Furthermore, the QTLs identified so far are limited to those
polymorphic between DBA/2 and C57BL/6 mice. Finally, this mapping study
considered only male mice, and we have provided evidence in two other
experiments for the existence of sex-specific, pain-relevant QTLs: a
locus (containing the -opioid receptor gene Oprd1)
statistically associated with basal nociception on the hot-plate test
in male mice only (Mogil et al., 1997a) and a locus on chromosome 8 mediating nonopioid stress-induced analgesia in female mice only (Mogil
et al., 1997b).
). In addition, the virtually identical AD50 values
between males of these strains should decrease the probability that the
QTLs identified will be highly relevant to males. Given the previous
success in identifying sex-specific QTLs of relevance to pain
processing, it seems reasonable to expect that sex differences in
morphine analgesia might also be due at least partially to the action
of separate genes in each sex. The identification of such genes and their protein products may shed further light on inconsistencies in the
existing literature and, more importantly, on mechanisms underlying the
variable actions of this clinically useful drug.
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Acknowledgments |
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We thank Brenda Edwards and her staff for their excellent care of animals and Dr. John K. Belknap for helpful discussions regarding the manuscript.
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Footnotes |
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Accepted for publication January 19, 1999.
Received for publication September 4, 1998.
1 This work was supported by National Institutes of Health Grants DA11394 and DE12735 (to J.S.M.) and Professional Staff Congress/City University of New York Grant 668648 (to B.K.).
Send reprint requests to: Dr. Benjamin Kest, Department of Psychology (4S-223), The College of Staten Island/City University of New York, 2800 Victory Blvd., Staten Island, NY 10314. E-mail: kest{at}postbox.csi.cuny.edu
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Abbreviations |
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AD50, half-maximal analgesic dose; %MPE, maximum possible effect; CI, confidence interval; NMDA, N-methyl-D-aspartate; SW, Swiss-Webster; QTL, quantitative trait locus.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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2-opioid receptors.
Pain
70:
267-277[Medline]. (Swiss Webster) and µ (ICR) responding mice.
Pharmacogenetics
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, males; 
,
females) represent strain mean percentages (±S.E.M.) of the %MPE at
each cumulative dose. Significant male > female sex differences
were found in AKR, C57BL/6, and SWR strains. Significant female > male sex differences were found in the CBA strain.