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. 2026 Apr 24;67(3):e70047. doi: 10.1002/em.70047

A Survey of DNA Damage in American Alligators ( Alligator mississippiensis ) in Florida

Haiyan Lu 1, Idoia Meaza 1, Rachel M Wise 1, James T F Wise 2, Sandra S Diven 1, Tayler J Croom‐Pérez 1, Jennifer H Toyoda 1, John Pierce Wise Sr 1,✉
PMCID: PMC13109689  PMID: 42033008

ABSTRACT

The scientific concept of One Environmental Health is a research strategy focused on the study of toxicants, aiming to incorporate human, wildlife, and ecosystem health to establish a more comprehensive understanding of health. A One Environmental Health approach, studying the responses of American alligators ( Alligator mississippiensis ) that are considered apex sentinel organisms to environmental toxicants, is crucial. Due to their long‐lived lifestyle on land and water, including a wide range of prey items in their diet and their ability to bioaccumulate metals, alligators serve as effective indicators of heavy metal pollution, which poses risks to aquatic ecosystems and human health. In this study, we examined DNA damage in American alligators from three locations in Florida using comet assay. We found alligators in Merritt Island National Wildlife Refuge had significantly higher DNA damage levels compared to those in Lake Apopka and Lake Woodruff. This trend was consistent across both sexes, with no observed sex differences. Similarly, DNA damage levels were significantly higher in both adult and juvenile alligators from Merritt Island compared to the other locations. Notably, juvenile alligators exhibited higher DNA damage than adults, with animals from Merritt Island exhibiting particularly elevated levels.

Keywords: American alligator, comet assay, DNA damage, metals

1. Introduction

One Health is an important and expanding research field that emphasizes the interconnectedness of human, animal, and environmental health. As both a research approach and a practical framework, One Health brings together expertise from fields such as medicine, veterinary science, biology, epidemiology, and toxicology, aiming to deepen our understanding of health through multidisciplinary collaboration (CDC 2024). One Environmental Health, a specific subset of One Health, is a research strategy focused on the study of toxicants (Pérez and Pierce Wise Sr. 2018). In a One Environmental Health approach, studying the responses of sentinel organisms to environmental toxicants is crucial, as these organisms can detect disturbances of various types—chemical, biological, physical, and more—at levels low enough to prevent negative impacts on humans, thus providing an early warning system for such hazards (National Research Council Committee on Animals as Monitors of Environmental Hazards 1991).

American alligators ( Alligator mississippiensis ) are positioned at the top of the food chain within their ecological niche, possess a relatively long lifespan, and are excellent sentinel organisms for studying how contaminants propagate through lower trophic levels and ultimately impact entire ecosystems (Scott et al. 2006; Pérez and Wise Sr. 2018). Exposure of wildlife to heavy metals and other chemicals has become a major concern in aquatic systems worldwide (Campbell et al. 2010). Due to their persistence in the environment, heavy metals are absorbed directly from abiotic sources (water, sediments, soil) or indirectly through prey or food, subsequently transferring through the food chain and biomagnifying, which makes top predators particularly vulnerable to their toxic effects (Saidon et al. 2024).

Pollution in Florida surface waters poses a significant threat to aquatic ecosystems, exposing alligators to various contaminants that cause DNA damage, ultimately compromising their genetic and epigenetic integrity and undermining their reproductive success and population viability (Rauschenberger et al. 2007; Gargee et al. 2011; Woodward et al. 2011; Guillette et al. 2016). Agricultural runoff, industrial discharge, and urban wastewater introduce pollutants such as pesticides, heavy metals, and industrial chemicals into water bodies in Florida (Winger et al. 1984; He et al. 2004; Worthington and Hutchinson 2008; Mitsch 2016; Li et al. 2022; Griffin et al. 2023). Lake Apopka has been contaminated for over 50 years with excessive nutrients and pesticides from nearby agricultural operations. The discharge of large amounts of organochlorine pesticides [e.g., dichloro‐diphenyl‐trichloroethane (DDT) and its derivatives] has been linked to disrupted sex hormone levels, abnormal gonadal development in newborn and juvenile alligators, increased juvenile mortality, and reduced clutch viability (Guillette et al. 1994, 1999, 2000; Rauschenberger et al. 2007). These studies revealed developmental and reproductive abnormalities linked to disrupted gene expression and endocrine signaling. Guillette et al. found alligators from contaminated Florida surface waters, such as Lake Apopka and Merritt Island, exhibited significant DNA methylome alterations, with numerous differential methylation regions linked to endocrine disruption, suggesting pollution's impact on epigenetic programming (Guillette et al. 2016). Milnes et al. discovered alligators from polluted Lake Apopka showed increased post‐hatching mortality and loss of sexually dimorphic gene expression, attributed to embryonic exposure to environmental contaminants, highlighting pollution's role in persistent developmental and genetic changes (Milnes et al. 2008). Another pollution problem in Florida's lakes and water bodies is eutrophication, driven by nutrient pollution from agricultural and urban fertilizer use, particularly nitrogen and phosphorus, which fosters harmful algal blooms that deplete oxygen, threaten aquatic life, and even cause DNA damage (Havens 2013; EPA 2024).

Metals are widespread environmental contaminants that pose a significant risk to aquatic ecosystems, with levels expected to rise due to climate change, which can release metals from sediments through acidification (de Orte et al. 2018). The American alligator's role as a keystone species makes it an ideal subject for studying the bioaccumulation and effects of heavy metals (Tellez and Merchant 2015; Wise et al. 2016). As an apex predator and keystone species, the alligator's exposure to metals can provide valuable insights into the health of its habitat and the potential risks to other species, including humans. Metals such as hexavalent chromium [Cr(VI)], arsenic, cadmium, lead, and mercury are of particular concern due to their toxicological effects and prevalence in contaminated environments (Tellez and Merchant 2015; Wise et al. 2016; Nilsen et al. 2019). Research has demonstrated the cytotoxic and genotoxic effects of Cr(VI) on alligator cells derived from scute tissue in a concentration‐dependent manner, indicating that these reptiles can be effective models for studying the impacts of environmental metal contamination (Wise et al. 2016). Additionally, alligators have been shown to bioaccumulate metals such as arsenic, cadmium, copper, iron, lead, selenium, zinc, and mercury, making them valuable bioindicators of metal pollution (Tellez and Merchant 2015; Nilsen et al. 2019). Additionally, studies have shown that alligators accumulate various heavy metals in their tissues, with significant site‐specific variations in metal concentrations, reflecting the degree of environmental contamination (Gunderson et al. 2016; Nilsen et al. 2019). For instance, alligators from metal‐polluted sites exhibit higher concentrations of metallothionein, a protein associated with metal detoxification, compared to those from less contaminated areas (Gunderson et al. 2016).

Exposure to genotoxic metals and metalloids like arsenic, cadmium, Cr(VI), lead, and nickel can cause significant DNA damage through mechanisms such as mutations, chromosomal instability, and impaired DNA repair, ultimately increasing cancer risk (Morales et al. 2016; Jomova et al. 2025; Meaza et al. 2024). Cr(VI) undergoes intracellular reduction to trivalent chromium [Cr(III)], generating various lesions, including oxidized bases, Cr–DNA adducts, DNA–DNA interstrand crosslinks and DNA strand breaks (Slade et al. 2005; Wise et al. 2008; Meaza et al. 2024). Nickel exposure has been shown to induce DNA damage by direct DNA binding and reactive oxygen species stimulation, while also repressing DNA repair systems such as nucleotide excision repair and base excision repair (Guo et al. 2019). Co‐exposure to multiple heavy metals, such as cadmium, cobalt, and lead, can result in more severe genotoxic effects than exposure to individual metals alone (Hengstler et al. 2003). This is due to potential interactions that inhibit DNA repair mechanisms, thereby increasing susceptibility to DNA damage (Hengstler et al. 2003). For instance, cobalt exposure, even at low levels, can significantly contribute to DNA single‐strand breaks when combined with cadmium and lead exposure (Hengstler et al. 2003).

The comet assay, also known as single‐cell gel electrophoresis, is a sensitive technique for detecting DNA strand breaks at the cellular level, making it a valuable tool in ecotoxicology (Kumaravel et al. 2009; Gajski et al. 2019). The assay assesses DNA damage by measuring the migration of DNA fragments during electrophoresis, where the extent of the “comet tail” indicates the extent of strand breaks (Kumaravel et al. 2009). Studies on fish and mollusks suggest the assay's sensitivity can be adapted for reptile cells, enabling DNA damage monitoring in crocodilian blood or tissue samples to assess the genotoxic effects of pollutants such as heavy metals (Mitchelmore et al. 1998; Ateeq et al. 2005; Bolognesi et al. 2019). Recent applications of the comet assay in Caiman crocodilus or Caiman latirostris hatchlings have revealed increased DNA damage in individuals exposed to metals or pesticides (Marrugo‐Negrete et al. 2019; Odetti et al. 2020).

Metal contamination in Florida's rivers has been increasing due to atmospheric emissions, surface discharges, and stormwater runoff from agriculture and urbanization, with some areas exceeding ecological thresholds for metals (Castro et al. 2013; Bielmyer‐Fraser et al. 2022). These rising metal loads, coupled with storm‐induced flooding, threaten waterway health and ecosystems (Tchounwou et al. 2012; Bielmyer‐Fraser et al. 2022). Given these trends and the potential for metals to cause DNA damage, we examined DNA damage in American alligators in Florida using the comet assay.

2. Materials and Methods

2.1. Sample Collection Area and Sample Process

We sampled alligators at three sites in Florida: Merritt Island National Wildlife Refuge (Merritt Island), Lake Apopka, and Lake Woodruff. Merritt Island, situated on a barrier island off Florida's east coast, encompasses the Kennedy Space Center. Most of the surrounding water of Merritt Island is brackish, with inland lakes and ditches creating a high diversity of prey items from various freshwater and saline habitats. The effects of rocket launches and potential contaminants released into the environment pose risks to wildlife in the refuge (Horai et al. 2014). Metal analyses of alligators captured at this site showed elevated concentrations of metals in tissues of juvenile and adult alligators compared with alligators from Lake Apopka and Lake Woodruff (Horai et al. 2014). Lake Apopka, situated in northwest Orange and Southeast Lake counties, is heavily polluted with organochlorine pesticides like DDT and its metabolites from industrial spills in the 1980s, as well as contaminants from wastewater treatment facilities and agricultural runoff (Guillette et al. 1999; Garrison et al. 2010). Lake Woodruff was chosen as the reference population because previous studies have demonstrated that organochlorine compounds at this site are lower than those at Lake Apopka and metal concentrations in alligator tissues are lower than those at Merritt Island (Guillette et al. 1999; Garrison et al. 2010; Horai et al. 2014).

ArcGIS mapping of alligator sampling locations was plotted using GPS coordinates based on the sample digital latitude and longitude with ArcGIS Pro version 3.4 [Environmental Systems Research Institute (ESRI), Redlands, CA]. The specific map used was the “Outdoor” Basemap provided in the program, which was developed for the program using data from Esri, TomTom, Garmin, FAO, NOAA, USGS, OpenStreetMap contributors, and the GIS User Community. The sampling locations are shown in Figure 1.

FIGURE 1.

FIGURE 1

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The maps of alligator sampling. American alligators were sampled in 3 areas within Florida. (A) Comprehensive map of all locations. (B) Sampling locations in Merritt Island. (C) Lake Apopka sampling locations. (D) Lake Woodruff sampling locations.

Animals were sampled under the State of Florida Fish and Wildlife Conservation Commission Permit (#SPGS‐16‐141R2) and the United States Department of the Interior Fish and Wildlife Service permit (# MI‐2019‐216R). Collection procedures were reviewed and approved by the University of Louisville and Kennedy Space Center's IACUC committee (protocol approval numbers UofL IACUC 18331 and GRD‐06‐044). 33 alligators were sampled in Merritt Island from November 3–5, 2018; 22 alligators were sampled in Lake Apopka from November 6–8, 2018; and 33 alligators were sampled in Lake Woodruff from November 9–10, 2018.

2.2. Blood Sample Collection and Alligator Measurements

After capturing large alligators using a hook and hand‐capturing small ones, blood samples were collected from alligators via the occipital sinus and immediately placed on ice (Myburgh et al. 2014). After sample collection and processing, each alligator was examined for any external injuries or behavioral abnormalities (e.g., lethargy) while determining head length, snout‐vent length, total length, sex, and age (Allsteadt and Lang 1995).

2.3. Isolation of Lymphocytes

Heparinized blood was gently mixed with an equal amount of PBS and deposited on 1.5 times amount of ficoll without mixing the layers. This mixture was centrifuged for 20 min at 1600 rpm. A micropipette tip was used to remove the lymphocytes, consisting of a whitish layer at the interface between the two layers. Lymphocytes were placed in a 15 mL tube with 5 mL of PBS. After centrifugation for 5 min at 1000 rpm, the supernatant was removed, and the pellet was washed twice more. Cells were counted using a Cellometer (Nexcelom Biocience, MA). The lymphocytes were brought to a cell density of 100,000 cells/mL.

2.4. Alkaline Comet Assay

The comet assay (i.e., single‐cell gel electrophoresis) is a method for measuring DNA damage in individual eukaryotic cells (Olive and Banáth 2006). In each set of comet assays, internal control and positive controls were included. Positive controls were prepared by exposing lymphocytes to 10 μM or 300 μM H₂O₂ for 30 min. The cell suspension was mixed with agarose (Trevigen, Cat# 4250–050‐02) at a ratio of 1:10 and spread evenly on comet slides (Trevigen, Cat# 4250‐200‐03). After solidification at 4°C, slides were immersed in a fresh comet lysis buffer (Trevigen, Cat# 4250‐050‐01) at 4°C for 30 min, followed by an unwinding incubation in a freshly prepared electrophoresis buffer (200 mM sodium hydroxide, 1 mM EDTA, pH > 13.0) for 1 h at room temperature. Electrophoresis was carried out at 21 V for 30 min in the freshly prepared electrophoresis buffer (pH > 13.0) at 4°C. Finally, slides were fixed in 70% ethanol and stained with SYBR green. All the steps described above were conducted under reduced light to prevent spurious DNA damage. Comet images were captured using an Olympus fluorescence microscope equipped with a SensiCam. Image analysis was carried out by Comet Assay IV software (Perceptive Inc., UK). Tail intensity, tail moment, and tail length were used as parameters to quantify DNA damage. One hundred randomly selected cells were analyzed for each sample.

2.5. Statistics

Statistical analyses were performed using GraphPad Prism 9.4.0. All data were expressed as the mean ± SEM (standard error of the mean). Statistical differences were assessed using a two‐tailed Student's unpaired t‐test or unpaired t‐test with Welch's correction for normal distribution parametric data, Mann–Whitney test for non‐normal distribution parametric data, respectively. When comparing two variables, a two‐way ANOVA was used for different sexes and ages. Statistical significance was p < 0.05, though values of p < 0.1 were noted. We include both levels of significance, as in some settings outcomes in studies that are meaningfully significant (i.e., p < 0.1) but that do not reach the statistical level of p < 0.05 are being presented as no different from controls, which is an incorrect conclusion (Wise et al. 2024). Thus, we provide the reader with multiple levels of significance to consider the outcomes.

3. Results

3.1. Alligator Characteristics From Three Lakes

A total of 33, 22, and 33 alligators were sampled from Merritt Island, Lake Apopka, and Lake Woodruff, respectively. The characteristics of American alligators from three locations, including age, sex, snout–vent length, and total length, were recorded and are presented in Table 1.

TABLE 1.

The characteristics of American alligators in Merritt Island, Lake Apopka, and Lake Woodruff.

Locations Sex Numbers Snout‐vent length (cm) Total length (cm)
Juvenile Adult Juvenile Adult Juvenile Adult
Merritt Island Female 13 4 69.88 ± 3.83 105.38 ± 6.84 141.85 ± 7.43 208.25 ± 11.67
Male 10 6 54.48 ± 2.78 132.08 ± 12.97 111.95 ± 5.41 259.83 ± 23.04
Lake Apopka Female 6 3 46.67 ± 7.25 136.00 ± 11.02 94.75 ± 14.13 237.67 ± 29.13
Male 5 8 44.46 ± 10.36 157.31 ± 10.18 91.80 ± 20.58 304.88 ± 19.38
Lake Woodruff Female 9 5 64.78 ± 6.71 126.70 ± 5.06 128.06 ± 12.87 248.80 ± 11.08
Male 14 5 61.39 ± 5.12 129.50 ± 13.72 122.79 ± 9.47 251.60 ± 25.65
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Note: Snout‐vent length and total length are expressed as the mean ± SEM.

3.2. DNA Damage in American Alligators From Three Locations

Using the alkaline comet assay, we assessed DNA breaks in alligators from Merritt Island, Lake Apopka, and Lake Woodruff. The average tail intensity values were 15.49, 8.58, and 8.61, respectively; the tail moment values were 16.53, 8.10, and 9.54, respectively; while the tail lengths were 86.39, 63.40, and 53.48, respectively. Alligators from Merritt Island exhibited significantly higher DNA damage levels than those from the other two lakes (Figure 2A,C,E).

FIGURE 2.

FIGURE 2

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DNA damage in American alligators in Merritt Island, Lake Apopka, and Lake Woodruff. DNA damage was measured in peripheral blood lymphocytes from alligators in three different locations using the comet assay. The data represent comet assay parameters (tail intensity, tail moment, or tail length) in peripheral blood lymphocytes of alligators from three locations. Each dot represents one individual alligator. Error bars = Standard error of the mean. *Significantly different between two different locations (**p < 0.01, ****p < 0.0001). (A) The tail intensity. (B) The ratio of tail intensity to snout‐vent length. (C) The tail moment. (D) The ratio of tail moment to snout‐vent length. (E) The tail length. (F) The ratio of tail length to snout‐vent length.

To explore the relationship between DNA damage and body size, we calculated the ratios of tail intensity to snout‐vent length, tail moment to snout‐vent length, and tail length to snout‐vent length for alligators from three locations. Even after normalizing for body size differences, the DNA damage levels in alligators from Merritt Island remained markedly higher than those from Lake Apopka and Lake Woodruff (Figure 2B,D,F).

3.3. Sex Differences in DNA Damage From Three Locations

To investigate potential sex differences in DNA damage, we examined the extent of DNA damage in female and male alligators from three locations. We found the DNA damage levels of female alligators and male alligators between the three locations were similar. In both male and female groups, DNA damage levels in alligators from Merritt Island were higher than those from the other two locations (Figure 3A,C,E). Specifically, the average tail intensity levels in female alligators in Merritt Island, Lake Apopka, and Lake Woodruff were 16.86, 7.07, and 11.75, while in male alligators, the average levels were 14.03, 9.62, and 6.30 across the three locations, respectively. The average tail moment levels in female alligators in Merritt Island, Lake Apopka, and Lake Woodruff were 18.12, 6.72, and 14.55, while in male alligators, the average levels were 14.85, 9.05, and 5.85 across the three locations, respectively. The average tail length levels in female alligators in Merritt Island, Lake Apopka, and Lake Woodruff were 87.02, 60.39, and 58.44, while in male alligators, the average levels were 85.71, 65.49, and 49.81 across the three locations, respectively. Even after normalizing for body size differences, the DNA damage levels in female and male alligators from the three locations were similar (Figure 3B,D,F).

FIGURE 3.

FIGURE 3

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Sex comparison in DNA damage in American alligators from Merritt Island, Lake Apopka, and Lake Woodruff. DNA damage was measured in peripheral blood lymphocytes from alligators in three different locations using the comet assay. Each dot represents one individual animal. Error bars = Standard error of the mean. *Significantly different between two different locations ( # p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (A) Sex comparison of the tail intensity. (B) Sex comparison of the ratio of tail intensity to snout‐vent length. (C) Sex comparison of the tail moment. (D) Sex comparison of the ratio of tail moment to snout‐vent length. (E) Sex comparison of the tail length. (F) Sex comparison of the ratio of tail length to snout‐vent length.

3.4. Age Differences in DNA Damage Among Alligators From Three Locations

To explore potential age‐related differences in DNA damage, we assessed DNA damage levels in adult and juvenile alligators from Merritt Island, Lake Apopka, and Lake Woodruff. When analyzing tail intensity, we observed that adult alligators from all three locations exhibited higher levels of DNA damage than juveniles (two‐way ANOVA, p = 0.0001) (Figure 4A). However, after normalizing for body size differences, these differences between adult and juvenile alligators disappeared (Figure 4B). Despite this, juvenile alligators from Merritt Island displayed significantly higher DNA damage levels than juveniles from the other two lakes, both before and after adjusting for body size differences. Similar patterns were observed for the tail moment. Adult alligators at all three sites showed higher DNA damage than juveniles (two‐way ANOVA, p < 0.01) (Figure 4C), but these differences were no longer evident following normalization for body size (Figure 4D). Nonetheless, juvenile alligators from Merritt Island exhibited significantly higher tail moment values than juveniles from the other two lakes, regardless of body size normalization. When analyzing tail length, adult alligators from Lake Apopka and Lake Woodruff exhibited significantly higher DNA damage levels compared to juveniles from the same lake (Figure 4E). However, after adjusting for body size differences, juvenile alligators exhibited significantly higher DNA damage levels compared to adults within the same lake (two‐way ANOVA, p < 0.0001) (Figure 4F). These findings suggest that DNA damage in juvenile alligators is influenced by both environmental factors and age, with Merritt Island juveniles showing consistently elevated damage levels relative to their counterparts in Woodruff and Apopka.

FIGURE 4.

FIGURE 4

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Age differences in DNA damage in American alligators from Merritt Island, Lake Apopka, and Lake Woodruff. The data show age comparisons of comet assay parameters (tail intensity, tail moment, or tail length) in peripheral blood lymphocytes of alligators from three locations. Each dot represents one individual animal. Error bars = Standard error of the mean. *Significantly different between two different locations or different ages in the same location ( # p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (A) Age comparison of the tail intensity. (B) Age comparison of the ratio of tail intensity to snout‐vent length. (C) Age comparison of the tail moment. (D) Age comparison of the ratio of tail moment to snout‐vent length. (E) Age comparison of the tail length. (F) Age comparison of the ratio of tail length to snout‐vent length.

3.5. Age Differences Among Different Sexes in DNA Damage From Three Locations

Since DNA damage levels differed between adult and juvenile alligators in the three locations, we further assessed DNA damage levels in adult and juvenile alligators in the female and male groups separately. For female alligators, when analyzing tail intensity, no significant differences in DNA damage levels were observed between adults and juveniles across the locations (Figure 5A,B). However, juvenile females from Merritt Island exhibited higher DNA damage levels than their counterparts from the other two locations, both before and after body size normalization. Similarly, analysis of tail moment showed no significant age‐related differences in DNA damage among female alligators across locations (Figure 5C,D). However, juvenile females from Merritt Island consistently displayed higher tail moment values than juvenile females from the other two locations, regardless of body size normalization. Regarding tail length, no significant differences were found between adult and juvenile females across the locations before standardization (Figure 5E). After adjusting for body size, juvenile females had significantly higher DNA damage levels than adult females within the same locations (two‐way ANOVA, p < 0.0001) (Figure 5F). For male alligators, tail intensity analysis showed that adult males had higher DNA damage levels than juveniles across the locations (two‐way ANOVA, p = 0.0005) (Figure 6A). However, these differences disappeared after body size standardization (Figure 6B). Despite this, male alligators—both adults and juveniles—from Merritt Island exhibited significantly higher DNA damage levels than those from the other locations, regardless of standardization. A similar pattern was observed for tail moment. Adult male alligators at all three locations exhibited higher DNA damage than juveniles (two‐way ANOVA, p < 0.01) (Figure 6C), but these differences were no longer evident after body size standardization (Figure 6D). Nonetheless, both adult and juvenile male alligators from Merritt Island consistently showed significantly higher tail moment values than males from the other locations, independent of standardization. Tail length analysis revealed no significant differences between adult and juvenile males before standardization (Figure 6E), but after standardization, juvenile males had significantly higher DNA damage levels than adults from the same locations (two‐way ANOVA, p < 0.0001) (Figure 6F). These results highlight consistently elevated DNA damage levels in both female and male juvenile alligators from Merritt Island compared to juveniles from the other locations.

FIGURE 5.

FIGURE 5

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Age‐related differences in DNA damage in female American alligators from Merritt Island, Lake Apopka, and Lake Woodruff. The data show age comparisons of comet assay parameters (tail intensity, tail moment, or tail length) in peripheral blood lymphocytes of female alligators from three locations. Each dot represents one individual animal. Error bars = Standard error of the mean. *Significantly different between two different locations or different ages in the same location ( # p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001). (A) Age comparison of the tail intensity. (B) Age comparison of the ratio of tail intensity to snout‐vent length. (C) Age comparison of the tail moment. (D) Age comparison of the ratio of tail moment to snout‐vent length. (E) Age comparison of the tail length. (F) Age comparison of the ratio of tail length to snout‐vent length.

FIGURE 6.

FIGURE 6

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Age‐related differences in DNA damage in male American alligators from Merritt Island, Lake Apopka, and Lake Woodruff. The data show age comparisons of comet assay parameters (tail intensity, tail moment, or tail length) in peripheral blood lymphocytes of male alligators from three locations. Each dot represents one individual animal. Error bars = Standard error of the mean. *Significantly different between two different locations or different ages in the same location ( # p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). (A) Age comparison of the tail intensity. (B) Age comparison of the ratio of tail intensity to snout‐vent length. (C) Age comparison of the tail moment. (D) Age comparison of the ratio of tail moment to snout‐vent length. (E) Age comparison of the tail length. (F) Age comparison of the ratio of tail length to snout‐vent length.

4. Discussion

Our study is the first to report DNA damage in American alligators and one of the few studies reporting DNA damage in a reptile species. We found alligators in polluted waters had higher DNA damage than those in relatively low‐polluted lakes, which is consistent with previous environmental surveys of DNA damage in Caiman crocodilus (Marrugo‐Negrete et al. 2019; Oliveira et al. 2021).

Our study found alligators in Merritt Island had significantly higher DNA damage than those in the other two lakes. It is known that metal exposure can cause DNA damage, and chemical analysis of juvenile and adult alligators captured in Merritt Island showed slightly elevated concentrations of metals (lithium, iron, nickel, strontium, indium, antimony, mercury, lead, bismuth) as well as PCBs and PBDEs in their tissues compared with alligators in the other two sampling sites likely due to space‐related activities as the Kennedy Space Center is located on the Merritt Island National Wildlife Refuge (Horai et al. 2014). Gunderson et al. found alligators from Merritt Island had significantly higher metallothionein than those in other lakes (Gunderson et al. 2016). Wise et al. found Cr(VI) is cytotoxic and genotoxic to American alligator cells (Wise et al. 2016). Our finding that alligators in Merritt Island had significantly higher DNA damage than those in the other two lakes may be caused by this relatively high metal exposure.

In both female and male alligators, we found alligators from Merritt Island had significantly higher DNA damage than those in other lakes. In terms of gender differences, we did not find gender differences in DNA damage in alligators, which is consistent with the study of Caiman crocodilus in Brazil (Oliveira et al. 2021). Oliveira et al. utilized comet assay to investigate DNA damage in 4 female and 4 male Caiman crocodilus from the Catalão Lake and 4 females and 3 males from the Manaus urban zone, and no sex‐specific differences were observed.

In the comparison of DNA damage between adult and juvenile alligators, we found juvenile alligators had higher DNA damage than adults. The DNA damage in juvenile alligators from Merritt Island was significantly higher than juvenile alligators from the other two lakes, suggesting that juvenile alligators may also be more exposed to metals and other toxicants. Horai et al. reported the concentrations of elements such as rubidium, titanium, manganese, copper, strontium, and barium in the livers of juvenile alligators from Merritt Island, Lake Apopka, and Lake Woodruff and were significantly higher than those in adults (Horai et al. 2014). Juveniles may be more sensitive to genotoxic stress because of their developing detoxification systems or higher metabolic rates. For instance, juvenile alligators from Merritt Island exhibited higher metallothionein concentrations and lower glutathione‐S‐transferase activity compared to those from the other two sites (Gunderson et al. 2016). The significantly higher DNA damage in juveniles from Merritt Island suggests unique environmental stressors or contaminants at this site. Metals such as copper and manganese can induce oxidative stress at elevated levels, leading to DNA damage (Pitié et al. 2006; Rehmani et al. 2017; Nicolai et al. 2021). The higher metal concentrations in juveniles may contribute to increased reactive oxygen species production, especially at Merritt Island. The specific combination of metals or co‐contaminants at Merritt Island may create synergistic effects, increasing the genotoxic burden.

The alkaline comet assay used in this study detects a spectrum of DNA lesions, including single‐strand breaks, alkali‐labile sites such as apurinic/apyrimidinic (AP) sites, and double‐strand breaks arising from closely opposed lesions or repair‐associated intermediates (Collins et al. 2023). Given the distinct contaminant profiles among collection sites, it is reasonable to speculate that the predominant forms of DNA damage in American alligators may differ in lesion type and mechanistic origin across locations. At Lake Apopka, long‐term exposure to organochlorine pesticides (e.g., DDT and dicofol) and nutrient‐driven eutrophication likely promotes oxidative stress through disruption of redox homeostasis and hypoxia‐reoxygenation cycles associated with algal blooms (Havens 2013; Mrema et al. 2013). These conditions are expected to promote oxidative base damage, alkali‐labile sites, and single‐strand breaks (Hegde et al. 2012; Hegde, Mantha, et al. 2012), among which AP site formation and single‐strand breaks are easily detectable under alkaline conditions, while oxidative base damage is difficult to detect (Muruzabal et al. 2021). In contrast, alligators from Merritt Island are more likely to be exposed to heavy metals and aerospace‐associated industrial contaminants and therefore may experience a greater burden of structurally complex DNA damage. This damage spectrum likely includes oxidative base damage, alkali‐labile sites, single‐strand breaks, and double‐strand breaks (Wise et al. 2008; Balali‐Mood et al. 2021; Ojo et al. 2022; Meaza et al. 2024), with alkali‐labile sites, single‐strand breaks, and double‐strand breaks primarily contributing to increased alkaline comet tail readings. These damages are caused by redox imbalance and by inhibition of DNA repair pathways induced by metals and aerospace‐related industrial pollutants (Balali‐Mood et al. 2021; Ojo et al. 2022; Meaza et al. 2024). At the reference site, Lake Woodruff, where industrial and agricultural inputs are comparatively limited, the observed DNA damage likely reflects baseline endogenous processes, including metabolic reactive oxygen species and normal DNA repair intermediates, and is therefore expected to be dominated by low‐level single‐strand breaks and transient alkali‐labile sites. Although the alkaline comet assay does not distinguish among specific lesion classes and has limited sensitivity for detecting oxidized bases without lesion‐specific enzyme modification, the observed patterns are broadly consistent with site‐specific contaminant exposure. Collectively, our findings suggest that Merritt Island populations primarily exhibit DNA strand breaks associated with heavy metal contamination, Lake Apopka populations show damage patterns consistent with oxidative stress linked to pesticide and eutrophication‐related exposures, and Lake Woodruff populations display predominantly low baseline levels of DNA damage. Future application of lesion‐specific approaches, including enzyme‐modified comet assays, γ‐H2A.X or RAD51 immunodetection, and assays for DNA‐protein crosslinks, would enable more refined characterization of the dominant damage types associated with each exposure scenario.

5. Conclusion

Our findings highlight location‐ and age‐dependent differences in DNA damage among alligator populations, with Merritt Island emerging as a hotspot of genotoxic stress. Elevated DNA damage (especially in juveniles) signals potential environmental health risks that merit closer scrutiny through expanded contaminant profiling and mechanistic studies.

Author Contributions

Haiyan Lu: conceptualization, methodology, validation, formal analysis, investigation, data curation, writing – original draft, writing – review and editing, visualization, supervision, project administration. Idoia Meaza: methodology, validation, formal analysis, investigation, data curation, writing – review and editing. Rachel M. Wise: methodology, validation, formal analysis, investigation, data curation, writing – review and editing. James T. F. Wise: methodology, data curation, writing – review and editing. Sandra S. Diven: methodology, validation, formal analysis, investigation, data curation, writing – review and editing. Tayler J. Croom‐Pérez: methodology, validation, formal analysis, investigation, data curation, writing – review and editing. Jennifer H. Toyoda: methodology, validation, formal analysis, investigation, data curation, writing – review and editing. John Pierce Wise Sr: conceptualization, methodology, validation, formal analysis, investigation, resources, data curation, writing – original draft, writing – review and editing, visualization, supervision, project administration, funding acquisition.

Funding

This work was supported by the National Institute of Environmental Health Sciences, R01ES016893, R35ES032876, T32ES011564 and La Caxia Foundation, LCF/BQ/EU22/11930060.

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

We would like to thank Russell Lowers, Benton Back, and Vincent Deem for field support in capturing alligators and collecting samples. We also thank Dr. Louis J. Guillette, Jr. for the inspiration to conduct this study, and the collaboration that preceded this work and developed the field protocols, though he passed before he could join us on this expedition. This work was conducted under a Wildlife Conservation Commission Permit (#SPGS‐16‐141R2) and the United States Department of the Interior Fish and Wildlife Service permit (# MI‐2019‐216R). Research reported in this publication was supported by the National Institute of Environmental Health Sciences of the National Institutes of Health under grant numbers R01ES016893 (J.P.W.), R35ES032876 (J.P.W.), and T32ES011564 (J.H.T., R.M.W., and J.P.W.). The project that gave rise to these results received the support of a fellowship from “La Caixa” Foundation (ID 100010434). The fellowship code is LCF/BQ/EU22/11930060 (IM). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

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