"Low-dose paroxetine exposure causes lifetime declines in male mouse body weight, reproduction and competitive ability as measured by the novel organismal performance assay", Gaukler et al 2015  (https://www.dropbox.com/s/ayzxe7xyhbieuvq/2015-gaukler.pdf / http://gen.lib.rus.ec/scimag/get.php?doi=10.1016%2Fj.ntt.2014.11.002)

An intriguing design: instead of using a handful of direct measures of animal health like mortality or cancer rates in a normal lab setting to examine the effect of an intervention on health, provide a competitive/dangerous somewhat-naturalistic setting and use the indirect measure of reproductive fitness; this may be more sensitive as the resource limits magnify impacts and fitness, while indirect, is affected by many or all negative effects and may be a very good composite effect to test.

(Fitness is determined, of course, by genotyping - one of the benefits of the fall in sequence costs is that you can just measure fitness directly even in commingled confusing populations. It's 2015 - genotype ALL THE THINGS!)

I think in this case most of the value is probably coming from the competitive/stressful/zero-sum environment provided the mice and not so much from the use of reproductive fitness, since a lot of the direct effects were statistically-significant as well as the decrease in reproductive fitness, but both are cool.

Excerpts:

"Paroxetine is a selective serotonin reuptake inhibitor (SSRI) that is currently available on the market and is suspected of causing congenital malformations in babies born to mothers who take the drug during the first trimester of pregnancy. We utilized organismal performance assays (OPAs), a novel toxicity assessment method, to assess the safety of paroxetine during pregnancy in a rodent model. OPAs utilize genetically diverse wild mice (Mus musculus) to evaluate competitive performance between experimental and control animals as they compete among each other for limited resources in semi-natural enclosures. Performance measures included reproductive success, male competitive ability and survivorship. Paroxetine-exposed males weighed 13% less, had 44% fewer offspring, dominated 53% fewer territories and experienced a 2.5-fold increased trend in mortality, when compared with controls. Paroxetine-exposed females had 65% fewer offspring early in the study, but rebounded at later time points, presumably, because they were no longer exposed to paroxetine. In cages, paroxetine-exposed breeders took 2.3 times longer to produce their first litter and pups of both sexes experienced reduced weight when compared with controls. Low-dose paroxetine-induced health declines detected in this study that were undetected in preclinical trials with doses 2.5-8 times higher than human therapeutic doses. These data indicate that OPAs detect phenotypic adversity and provide unique information that could be useful towards safety testing during pharmaceutical development.

Like paroxetine, many medications once considered safe are found to cause unacceptable health consequences after public release. On average, 73% of pharmaceuticals fail during clinical trials (Lipsky and Sharp, 2001) and 10% of FDA approved pharmaceuticals are recalled after market release (Schuster et al., 2005), despite the 12-15 years of research and $1.4 billion average cost associated with each drug during development (Miller, 2012). One cause of the high pharmaceutical failure rate is the inability of current toxicity assessment methods to detect cryptic, or otherwise undetectable, adversities during preclinical trials, particularly those present at doses near therapeutic levels and/or those occurring at low incidences.
We have developed a novel toxicity assessment research method that may be useful during preclinical assessment, known as the organismal performance assay (OPA). In several instances, OPAs have proven capable of detecting mammalian health declines that were not visible to standard laboratory methodologies. OPAs utilize genetically diverse wild-derived mice (Mus musculus) that compete among each other for limited resources in semi-natural enclosures, which allows for direct competition between treatment and control individuals. OPAs assess the quality of individual mice (organisms) in terms of Darwinian fitness (i.e., reproductive success) and components leading to fitness (i.e., survivorship and male competitive ability), while residing in a naturalistic environment where the stresses that have shaped their evolutionary history are present. The sensitivity of the OPA derives from the fact that wild mice under social competition allows small changes in behavior or physiological performance and otherwise cryptic effects of toxicity to be manifested as measurable negative outcomes; such as relegation to inferior habitat and reduced reproduction and survival. Consequently, any degradation in almost any physiological system caused by a treatment will be detectable by the inability of mice to perform comparable to controls with whom they compete and will be revealed in OPA endpoint measures.
OPAs have previously been used to quantify the adverse effects of sibling-level and cousin-level inbreeding (Meagher et al., 2000; Ilmonen et al., 2008), harboring a selfish gene (Carroll et al., 2004) and recently, they were the first assay to reveal the adverse effects of added sugar consumption at human-relevant levels (Ruff et al., 2013). In all of these studies, OPAs found substantial deleterious effects that were missed by current methodologies.

- Meagher S, Penn DJ, Potts WK. "Male-male competition magnifies inbreeding depression in wild house mice" http://www.pnas.org/content/97/7/3324.full . Proc Natl Acad Sci U S A 2000;97:3324-9. http://dx.doi.org/10.1073/pnas.97.7.3324.
- Ilmonen P, Penn DJ, Damjanovich K, Clarke J, Lamborn D, Morrison L, et al. "Experimental infection magnifies inbreeding depression in house mice" http://www.researchgate.net/profile/Dustin_Penn/publication/5540638_Experimental_infection_magnifies_inbreeding_depression_in_house_mice/links/02e7e524fd3c729eb9000000.pdf . J Evol Biol 2008;21:834-41. http://dx.doi.org/10.1111/j.1420-9101.2008.01510.x.
- Carroll LS, Meagher S, Morrison L, Penn DJ, Potts WK. "Fitness effects of a selfish gene (the Mus t complex) are revealed in an ecological context" http://www.researchgate.net/profile/Shawn_Meagher/publication/8442534_Fitness_effects_of_a_selfish_gene_(the_Mus_t_complex)_are_revealed_in_an_ecological_context/links/02bfe513e70f7b104d000000.pdf . Evolution 2004;58:1318-28. http://dx.doi.org/10.1554/03-544. - Ruff JS, Suchy AK, Hugentobler SA, Sosa MM, Schwartz BL, Morrison LC, et al. "Human-relevant levels of added sugar consumption increase female mortality and lower male fitness in mice" http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3775329/. Nat Commun 2013;4. http://dx.doi.org/10.1038/ncomms3245.

Genetic diversity of this mice colony was assessed in the 11th generation and found to be comparable to wild populations (Cunningham et al., 2013).

Dosing was achieved by incorporating 7.5 g paroxetine (GSK, molecular formula: C 19 H 20 FNO 3 · HCl) into 50 kg of rodent chow (TD.130006; Harlan Teklad, Madison, WI). Mice consuming an average of 3 g of food per day and weigh 20 g will ingest 0.45 mg per day or 22.5 mg/kg/day. Using a standard metabolic rate conversion factor, this is equivalent to a human dose of 1.82 mg/kg/day, or a daily dose of 109.20 mg, assuming the average human weighs 60 kg (Reagan-Shaw et al., 2008). Given that paroxetine is prescribed in the range of 20-60 mg/day (Dunner and Dunbar, 1992; GSK, 2013), our dose is 1.82-fold higher than human therapeutic doses, yet lower than doses used in previous animal studies (Coleman et al., 1999; Rayburn et al., 2000).

Sixty breeder pairs were selected for this experiment; 20 pairs were exposed to paroxetine while the remainder served as controls. The asymmetry in cage number is due to the production of additional control animals for another study. Prior to breeding, animals were individually housed and provided with their respective diets. To maximize the chances of detecting adverse effects, both females and males were exposed to paroxetine prior to breeding (females were exposed to paroxetine eight days prior and males five days). Exposure to paroxetine continued when breeders were paired. By exposing both females and males, we were consistent with previous rodent studies (Coleman et al., 1999; Rayburn et al., 2000; El-gaafarawi et al., 2005), and it is likely that any adverse effects detected in the progeny are due to in utero exposure because birth defects have been observed in humans when women are prescribed paroxetine during pregnancy (Diav-Citrin et al., 2008).

Enclosures have previously been described in Ruff et al. (2013). Briefly, the indoor enclosures are approximately 30 m 2 and consist of two types of territories, optimal (n = 4) and suboptimal (n = 2). Each optimal territory contained a defendable box with multiple dark nesting sites and direct access to food. Suboptimal territories contained two nesting boxes exposed to light and had indirect access to food. Territories were separated by hardware mesh that is easily climbed, but added an element of spatial complexity (Fig. S1).
Five independent OPA populations were established and maintained for 28 weeks. OPA populations consisted of eight males and 14-16 females, for a total of 116 animals (40 males, 76 females); these animals are referred to as population founders. Half of the individuals of each sex were paroxetine-exposed while the remainder served as controls...Enclosure space and population size created a population density within the range observed in the wild (Sage, 1981).

Reproductive success of founders was determined by removing and genotyping all offspring in enclosures. Every five weeks, research personnel conducted a "pup sweep," in which all offspring were removed, sacrificed and had a tissue sample collected for genetic analyses. As the gestation period of mice is three weeks, the first pup sweep occurred on week eight, then, every sweep following occurred on five-week intervals; this sweep schedule prevented offspring from reaching sexual maturity in enclosures. A total of 872 samples were collected with an average of 174.4 ± 17.1 (M ± SEM) offspring per population.

2.6. Survivorship
Non-invasive health checks were performed daily and extensive enclosure checks every five weeks during pup sweeps. Extensive enclosure checks were limited to avoid disrupting territoriality that increases the rate of infanticide. When deceased founders were observed, they were removed from the enclosures. The date of death was estimated upon the condition of the corpse. Severely decomposed founders were given a date half way between the date the individual was found and the last date the PIT tag of that individual was recorded.

Paroxetine-exposed breeders took 2.3 times longer to produce their first litter when compared to controls (PH; χ 2 = 3.98, p b 0.05; Fig. 2), however; litter size was not affected by treatment (GLMM; z = −0.58, p = 0.56). Control breeders produced an average of 4.54 (SEM +0.45, − 0.41) pups in their first litter, while paroxetine-exposed breeders produced 4.09 (+ 0.83, − 0.75) pups in their first litter...Offspring from paroxetine-exposed breeders weighed less at weaning than offspring from control breeders. Paroxetine-exposed female offspring weighed 16% less than controls with an average of 8.81 g (SEM ± 0.52), whereas control female offspring weighed 10.77 g (± 0.31)

Paroxetine-exposed males weighed 18.42 g (± 0.87) and controls weighed 21.75 g (±0.90)

Female reproductive success was hindered by paroxetine exposure (GLMM; z = − 5.03, p b 0.0001; Fig. 5A) at week eight (model intercept), where mean reproduction of paroxetine-exposed females was 65% less than controls. Paroxetine-exposed females had an average of 10.68 (+1.44, −1.26) offspring per population, while control females had an average of 20.16 (+1.63, −1.51) offspring per population.

Paroxetine exposure also negatively affected male reproductive success as measured by male offspring, where paroxetine-exposed males had 44% fewer offspring than controls (GLMM; z = −2.72, p b 0.01; Fig. 5B).

Male competitive ability was adversely impacted by paroxetine-exposure, where paroxetine males occupied 53% fewer territories than controls. At week three (model intercept) control males occupied 47% of the territories, while paroxetine-exposed males occupied 22% territories (GLMM; z = −4.11, p b 0.0001; Fig. 6); leaving 31% of the territories undefended. The percent of undefended territories is not unusual because 2/6 (or 33%) were suboptimal and often difficult to defend.

No differences were detected in mortality between paroxetine-exposed and control females (PH; χ 2 = 0.66, p = 0.42; Fig. 7A). Female mortality rates did not differ in replicate populations (PH; χ 2 = 3.51, p = 0.48), nor was there a difference in the effect of treatment among populations (PH; χ 2 = 3.35, p = 0.50). A marginally significant trend was detected in which male mortality was increased approximately 2.5-fold by paroxetine exposure (PH; χ 2 = 3.27, p = 0.07; Fig. 7B).

Paroxetine-exposed males experienced reduced reproductive success, producing 44% fewer offspring than controls. Previous OPA studies reveal that dominant males sire N80% of offspring within enclosures (Carroll et al., 2004) and therefore differences in competitive ability likely explain a large portion of the differential reproduction, though other mechanisms are possible. A trend was observed in which paroxetine-exposed males suffered greater mortality and thus even if not significant, it does contribute to the reduced reproduction. Another possibility is that paroxetine has direct negative impacts on the male reproductive system. Paroxetine exposure has been shown to reduce sperm count (Baldwin et al., 1989; El-gaafarawi et al., 2005) and to increase sperm abnormalities in rats when exposed at a human therapeutic dose (Elgaafarawi et al., 2005). Additionally, hormones important for spermatogenesis (Kovacs, 2012) have also been altered by paroxetine exposure in rats (El-gaafarawi et al., 2005). These are all potential causes that could lead to reduced reproductive success, but additional experiments are needed to decipher the mechanisms causing this phenotype.

Since OPA studies generate the same outputs, relative fitness can be compared between treatments and for example, results from this study indicate that paroxetine-exposed male fitness (i.e., reproductive success) is ~ 25% less than the inbred males born to parents of first cousins (Fig. 8)."