Rodents cause devastating damage to both agriculture and ecosystems worldwide. Invasive rodents are commonly found on islands, historically free of these animals, and have significant negative impacts on both native plant and animal species. Rodents are exceptionally well adapted to their environments and therefore, quite challenging to control. Current control strategies often include large scale applications of toxicants, which have potential adverse effects on non-target wildlife. In island ecosystems, these adverse effects are a major hurdle to eradication efforts. The time and financial resources required to minimizing risks to non-target species and performing post-eradication exposure monitoring can limit the number of islands from which rodents are successfully eradicated. Therefore, the development of new species-specific rodenticides would be a valuable advancement in the effort to control these pest species, especially for island eradications. To that end, USDA Wildlife Services is investigating the use of interfering RNA (RNAi) as a novel way to control rodent species. RNAi reduces the amount of a specific protein that is made by a cell. This is done through post-transcriptional gene-silencing. The RNAi pathway is initiated when a small section of double stranded RNA is introduced into the cytoplasm of a cell. This double stranded RNA comprises a guide strand and a passenger strand. In the first step of the RNAi pathway, the foreign RNA is incorporated into an enzyme complex called RNA-induced silencing complex (RISC) at which time the passenger strand is degraded. The RISC/RNA complex then finds the complementary mRNA made by the cell and binds to it. Subsequently, the RISC complex degrades the complementary mRNA. This breaking down of the mRNA prevents the synthesis of the corresponding protein. The reduction in protein synthesis is the benchmark of RNAi and is how it will be used to elucidate lethal physiological changes in pest species. The species specificity of RNAi depends on the selection of portions of genes that are unique to the target animal species. By screening the rodent genome and comparing sequences of rodent genes to non-target species, we can choose sections of genes that are present in the pest species and absent in the non-target species. Previous research has established guidelines for both the nucleic acid composition of RNAi sequences and their location in the corresponding mRNA that facilitate maximum inhibition of protein synthesis while maintaining species specificity. Therefore, unlike current rodenticides, if non-target species consume the RNAi they will not be affected. This specificity could allow for the eradication of rodents from islands that have historically not been feasible. For RNAi to be useful as a rodenticide, it will likely have to be formulated in a bait for oral consumption. This is a significant hurdle: most RNAi based therapeutics for human use are formulated for intravenous or subcutaneous injection. Like all rodenticide baits, an RNAi-based bait will have to be stable in a wide range of environmental conditions and have a long shelf life. However, once consumed by the target animal, RNAi-based baits will have to be significantly more complex than traditional toxicant baits. The double stranded segments of RNA will have to be protected from the changing pH of the digestive tract and absorbed into the systemic circulation while maintaining viability. Once in circulation, RNAi molecules must be delivered to the target tissues at concentrations high enough to elucidate a physiological response. Advances in bioengineering have given researchers products that both hide RNAi inside stable exterior shells and direct these carrier molecules to the site of action. These cutting-edge technologies make the development of a RNAi-based rodent bait a feasible option. The use of RNAi for rodent control shows promise because of its species specificity and low non-target impact. RNAi sequences are selected to sections of the rodent genome that are significantly different from non-target species genes and therefore do not bind to and degrade the non-target mRNA. Formulating RNAi into oral baits presents challenges but recent advances in bioengineering have led to the development of mechanisms for delivery useful for this application of RNAi. RNAi-based baits will be a great benefit to efforts to eradicate rodents off islands because they will reduce the time and funding necessary to mitigate risks to non-target species and the environment.

In 1983, Savarie and colleagues did an extensive study of the toxicity of para-aminopropiophenone (PAPP) in multiple species of mammals and birds (Savarie et al. 1983). They tested 15 species and found that PAPP was toxic to coyotes: the LD50 for coyotes via oral gavage in cod liver oil was 5.6 mg/kg. However, they also found that coyotes (and numerous other species) vomited after consuming PAPP and this vomiting likely decreased the efficacy of PAPP. They concluded that the induction of vomiting by PAPP must be addressed before it could be effective as a chemical to control coyotes (Savarie et al. 1983). Huo Ping Pan further investigated the effects of PAPP to coyotes in studies compiled in an internal report [Pan, unpublished report, National Wildlife Research Center (NWRC)]. In the study of Savarie et al. (1983), coyotes that consumed 150 mg PAPP in beef tallow baits vomited and recovered. Pan hypothesized that if the dose of PAPP was increased slightly, coyotes may succumb to PAPP without vomiting. He dosed coyotes with 200, 300, or 400 mg PAPP; all the coyotes except two at the 200 mg dose succumbed to the effects of PAPP. These two animals both lapsed into unconsciousness but had to be euthanized. One of these two animals and one animal at the 300 mg dose “slightly” vomited. Pan went on to test a dose of 350 mg more thoroughly. Nine of ten coyotes died within 2.5 hours of dosing and one survived. None of the ten coyotes dosed with 350 mg of PAPP vomited (Pan, unpublished report, NWRC). By increasing the dose of PAPP, Pan was able to overcome the problem of emesis that Savarie et al. (1983) reported. With the data generated from these studies, Pan and colleagues concluded that work with PAPP should continue, as they thought that it could be developed as a “superior substitute for 1080 as coyote control toxicant.” PAPP has been registered in New Zealand for the control of stoats and feral cats and in Australia for the control of wild dogs and foxes. The USDA APHIS Wildlife Services National Wildlife Research Center (NWRC) has an interest in the development of PAPP for coyote control. NWRC investigated the efficacy of PAPP as a means of lethal control in coyotes using multiple routes of administration. In initial studies, animals were orally gavaged with PAPP in an inert carrier to determine LD50 values and test LD100 doses. When used on the landscape a toxicant must be efficacious under varied conditions such as full or empty stomach. Therefore, after the initial trials, studies were done to determine the efficacy of PAPP in coyotes when the animals consumed PAPP with other food. Coyotes were also offered PAPP in meatballs and allowed to freely consume the food bait. In further trials, NWRC researchers tested the effectiveness of two different doses of PAPP capsules (880 mg and 400 mg) placed in a spring-loaded ejector device (SLED) to lethally control coyotes. Both doses were lethal to all coyotes tested, supporting further research into the development of PAPP as a tool to control coyotes.

Liver microsomes are used extensively in human pharmaceutical development to study the metabolism of compounds of interest; however, they are rarely part of the development of toxicants for the control of wildlife. Liver microsome samples from wildlife of interest would likely be harvested in the field without access to laboratory perfusion equipment. Therefore, the metabolic activity of microsomes from perfused and non-perfused livers was compared. There was no significant difference in diphacinone metabolism by liver microsomes from both perfused and non-perfused Wistar rat livers, although chlorophacinone metabolism was significantly different. There are often significant differences in metabolism between species that can be utilized to increase toxicant specificity. In this study bobwhite quail liver microsomes metabolized more diphacinone and chlorophacinone than both Wistar and brown rats. This information can be used during toxicant development to help determine the most sensitive species to toxicants of interest. Additionally, the effect of incubation time on toxicant metabolism was examined to determine optimal experimental conditions. The data from these experiments support the use of liver microsomes as a tool to be used during toxicant development to provide information that can be incorporated into whole animal studies.

The acute oral toxicity of the anticoagulant rodenticide diphacinone was found to be about 20 times greater to American kestrels (LD50=97 mg/kg) than to northern bobwhite (LD50=2,014 mg/kg). Several precise and sensitive clotting assays (prothrombin time, Russell’s Viper venom time, thrombin clotting time) were adapted for use in these species, and this combination of assays is recommended to detect effects of diphacinone and other rodenticides on coagulation. Oral administration of diphacinone over a range of doses (sublethal to the extrapolated LD15) prolonged prothrombin time and Russell’s Viper venom time within 24 to 48 hrs post-exposure. Prolongation of in vitro clotting time reflects impaired coagulation complex activity and was detected before or at the onset of overt signs of toxicity and lethality. These data will assist in the development of a pharmacodynamic model to assess and predict rodenticide toxicity to non-target avian species.

A seminal question in wildlife toxicology is whether exposure to an environmental contaminant, in particular a second-generation anticoagulant rodenticide, can evoke subtle long lasting effects on body condition, physiological function and survival. Many reports indicate that non-target predators often carry residues of several rodenticides, which is indicative of multiple exposures. An often-cited study in laboratory rats demonstrated that exposure to the second-generation anticoagulant rodenticide brodifacoum prolongs blood clotting time for a few days, but weeks later when rats were re-exposed to the first-generation anticoagulant rodenticide warfarin, coagulopathy was more pronounced in brodifacoum-treated rats than naïve rats exposed to warfarin. To further investigate this phenomenon, American kestrels were fed environmentally realistic doses of chlorophacinone or brodifacoum for a week, and following a week-long recovery period, birds were then challenged with a low-level dietary dose of chlorophacinone. In the present study, neither hematocrit nor clotting time (prothrombin time, Russell’s viper venom time) were differentially affected in sequentially exposed kestrels compared to naïve birds fed low-level dietary dose of chlorophacinone. While the present findings do not reveal lasting effects of anticoagulant exposure on blood clotting ability, findings in laboratory rats and other species have demonstrated such effects on blood clotting, and even other molecular pathways associated with immune function and xenobiotic metabolism. Additional studies using an environmentally realistic route of exposure and dose are underway to further test this hypothesis.

Regulatory changes in the use of some second-generation anticoagulant rodenticides in parts of North America may result in expanded use of first-generation anticoagulant rodenticides (FGARs). Recent toxicological studies with captive raptors have demonstrated that these species are considerably more sensitive to the FGAR diphacinone than traditional avian wildlife test species (mallard, bobwhite). We have now examined the toxicity of the FGAR chlorophacinone (CPN) to American kestrels fed rat tissue mechanically amended with CPN, or rat tissue containing biologically-incorporated CPN, for 7 days. Nominal CPN concentrations in these diets were 0.15, 0.75, and 1.5 µg/g food wet weight, and actual CPN concentration in diets were analytically verified as being close to target values. Food intake was consistent among groups, body weight fluctuated by less than 6%, exposure and adverse effects were generally dose-dependent, and there were no dramatic differences in toxicity between mechanically-amended and biologically-incorporated CPN diets. Using benchmark dose statistical methods, toxicity reference values at which clotting times were prolonged in 50% of the kestrels was estimated to be about 80 µg CPN consumed/kg body weight-day for prothrombin time and 40 µg CPN/kg body weight-day for Russell’s viper venom time. Based upon carcass CPN residues reported in rodents from field baiting studies, empirical measures of food consumption in kestrels, and dietary-based toxicity reference values derived from the 7-day exposure scenario, some free-ranging raptors consuming CPN-exposed prey might exhibit coagulopathy and hemorrhage. These sublethal responses associated with exposure to environmentally realistic concentrations of CPN could compromise survival of exposed birds.

New regulatory restrictions have been placed on the use of some second-generation anticoagulant rodenticides in the United States, and in some situations this action may be offset by expanded use of first-generation compounds. We have recently conducted several studies with captive adult American kestrels and eastern screech-owls examining the toxicity of diphacinone (DPN) using both acute oral and short-term dietary exposure regimens. Diphacinone evoked overt signs of intoxication and lethality in these raptors at exposure doses that were 20 to 30 times lower than reported for traditionally used wildlife test species (mallard and northern bobwhite). Sublethal exposure of kestrels and owls resulted in prolonged clotting time, reduced hematocrit, and/or gross and histological evidence of hemorrhage at daily doses as low as 0.16 mg DPN/kg body weight. Findings also demonstrated that DPN was far more potent in short-term 7-day dietary studies than in single-day acute oral exposure studies. Incorporating these kestrel and owl data into deterministic and probabilistic risk assessments indicated that the risks associated with DPN exposure for raptors are far greater than predicted in analyses using data from mallards and bobwhite. These findings can assist natural resource managers in weighing the costs and benefits of anticoagulant rodenticide use in pest control and eradication programs.

Weight-loss interventions generally improve lipid profiles and reduce cardiovascular disease risk, but effects are variable and may depend on genetic factors. We performed a genetic association analysis of data from 2,993 participants in the Diabetes Prevention Program to test the hypotheses that a genetic risk score (GRS) based on deleterious alleles at 32 lipid-associated single-nucleotide polymorphisms modifies the effects of lifestyle and/or metformin interventions on lipid levels and nuclear magnetic resonance (NMR) lipoprotein subfraction size and number. Twenty-three loci previously associated with fasting LDL-C, HDL-C, or triglycerides replicated (P = 0.04-1 × 10(-17)). Except for total HDL particles (r = -0.03, P = 0.26), all components of the lipid profile correlated with the GRS (partial |r| = 0.07-0.17, P = 5 × 10(-5)-1 10(-19)). The GRS was associated with higher baseline-adjusted 1-year LDL cholesterol levels (β = +0.87, SEE ± 0.22 mg/dl/allele, P = 8 × 10(-5), P(interaction) = 0.02) in the lifestyle intervention group, but not in the placebo (β = +0.20, SEE ± 0.22 mg/dl/allele, P = 0.35) or metformin (β = -0.03, SEE ± 0.22 mg/dl/allele, P = 0.90; P(interaction) = 0.64) groups. Similarly, a higher GRS predicted a greater number of baseline-adjusted small LDL particles at 1 year in the lifestyle intervention arm (β = +0.30, SEE ± 0.012 ln nmol/L/allele, P = 0.01, P(interaction) = 0.01) but not in the placebo (β = -0.002, SEE ± 0.008 ln nmol/L/allele, P = 0.74) or metformin (β = +0.013, SEE ± 0.008 nmol/L/allele, P = 0.12; P(interaction) = 0.24) groups. Our findings suggest that a high genetic burden confers an adverse lipid profile and predicts attenuated response in LDL-C levels and small LDL particle number to dietary and physical activity interventions aimed at weight loss.

Sex hormone binding globulin (SHBG) levels are reported to be inversely associated with diabetes risk. It is unknown whether diabetes prevention interventions increase SHBG and whether resultant changes in SHBG affect diabetes risk. The purpose of this analysis was to determine whether intensive lifestyle intervention (ILS) or metformin changed circulating SHBG and if resultant changes influenced diabetes risk in the Diabetes Prevention Program (DPP). This is a secondary analysis from the DPP (1996-2001), a randomized trial of ILS or metformin versus placebo on diabetes risk over a mean follow-up of 3.2 years. The DPP was conducted across 27 academic study centers in the USA. Men, premenopausal and postmenopausal women without hormone use in the DPP were evaluated. The DPP included overweight/obese persons with elevated fasting glucose and impaired glucose tolerance. Main outcomes measures were changes in SHBG levels at 1 year and risk of diabetes over 3 years. ILS resulted in significantly higher increases (postmenopausal women: p<0.01) or smaller decrements (men: p<0.05; premenopausal women: p<0.01) in SHBG compared with placebo or metformin. Changes in SHBG were primarily attributable to changes in adiposity. There were no consistent associations of change in SHBG with the risk of diabetes by treatment arm or participant group. Lifestyle intervention may be associated with favorable changes in circulating SHBG, which is largely due to changes in adiposity. Changes in circulating SHBG do not independently predict reductions in diabetes incidence.

Objective

To determine whether metformin or lifestyle modification can lower rates of all-cause and cause-specific mortality in the Diabetes Prevention Program and Diabetes Prevention Program Outcomes Study.

Research design and methods

From 1996 to 1999, 3,234 adults at high risk for type 2 diabetes were randomized to an intensive lifestyle intervention, masked metformin, or placebo. Placebo and lifestyle interventions stopped in 2001, and a modified lifestyle program was offered to everyone, but unmasked study metformin continued in those originally randomized. Causes of deaths through 31 December 2018 were adjudicated by blinded reviews. All-cause and cause-specific mortality hazard ratios (HRs) were estimated from Cox proportional hazards regression models and Fine-Gray models, respectively.

Results

Over a median of 21 years (interquartile range 20-21), 453 participants died. Cancer was the leading cause of death (n = 170), followed by cardiovascular disease (n = 131). Compared with placebo, metformin did not influence mortality from all causes (HR 0.99 [95% CI 0.79, 1.25]), cancer (HR 1.04 [95% CI 0.72, 1.52]), or cardiovascular disease (HR 1.08 [95% CI 0.70, 1.66]). Similarly, lifestyle modification did not impact all-cause (HR 1.02 [95% CI 0.81, 1.28]), cancer (HR 1.07 [95% CI 0.74, 1.55]), or cardiovascular disease (HR 1.18 [95% CI 0.77, 1.81]) mortality. Analyses adjusted for diabetes status and duration, BMI, cumulative glycemic exposure, and cardiovascular risks yielded results similar to those for all-cause mortality.

Conclusions

Cancer was the leading cause of mortality among adults at high risk for type 2 diabetes. Although metformin and lifestyle modification prevented diabetes, neither strategy reduced all-cause, cancer, or cardiovascular mortality rates.