Kevin CliverKevin CliverAbnormal Bone composition in Female Juvenile Abnormal Bone composition in Female Juvenile American Alligators From a Pesticide-Polluted American Alligators From a Pesticide-Polluted

Lake (Lake Apopka, Florida)Lake (Lake Apopka, Florida)

Lind, P. Monica; Milnes, Matthew R.; Lind, P. Monica; Milnes, Matthew R.; Lundberg, Rebecca; Bermudez, Lundberg, Rebecca; Bermudez,

Dieldrich; Orberg, Jan; Guillette Jr., Louis Dieldrich; Orberg, Jan; Guillette Jr., Louis J. J. Environmental Health PerspectivesEnvironmental Health Perspectives. . Vol. 112 (3). March Vol. 112 (3). March 20042004. 359-362 . 359-362

BackgroundBackground

• Test subject is the American alligatorTest subject is the American alligator• Test sights are at the pesticide polluted Lake Test sights are at the pesticide polluted Lake

Apopka and Lake Woodruff located in Florida.Apopka and Lake Woodruff located in Florida.• 1980 – Industrial pesticide spill in Lake 1980 – Industrial pesticide spill in Lake

Apopka contaminating it with endocrine active Apopka contaminating it with endocrine active pesticides (DDT) and dicofol know as pesticides (DDT) and dicofol know as endocrine disrupting chemicals (EDC’s).endocrine disrupting chemicals (EDC’s).

• Estrogens exhibit dramatic influences on Estrogens exhibit dramatic influences on bones and are essential for bone homeostasis. bones and are essential for bone homeostasis.

• An increase in estrogenic levels increase the An increase in estrogenic levels increase the amount of bone tissue, and visa versa.amount of bone tissue, and visa versa.

Hypothesis and aims of the Hypothesis and aims of the studystudy• Their hypothesis was that the contaminants Their hypothesis was that the contaminants

present in the lake inhibit the natural and present in the lake inhibit the natural and continuous resorption of bone tissue by the continuous resorption of bone tissue by the anti-androgenic and estrogenic actions of the anti-androgenic and estrogenic actions of the pesticides, resulting in and increased bone pesticides, resulting in and increased bone mass when compared to alligators from mass when compared to alligators from unpolluted lakes.unpolluted lakes.

• Because 17Because 17ß-estroanol and testosterone play ß-estroanol and testosterone play an important role in bone tissue homeostasis, an important role in bone tissue homeostasis, the aim of the study was to examine possible the aim of the study was to examine possible effects on the skeleton of female juvenile effects on the skeleton of female juvenile alligators from a polluted lake with EDC’s.alligators from a polluted lake with EDC’s.

Materials and MethodsMaterials and Methods• 16 juvenile female alligators of equal age 16 juvenile female alligators of equal age

and size were collected by hand from the and size were collected by hand from the two lakes; contaminated Lake Apopka, and two lakes; contaminated Lake Apopka, and a reference lake Woodruff.a reference lake Woodruff.

• A lethal dose of sodium pentobarbital was A lethal dose of sodium pentobarbital was given to the alligators.given to the alligators.

• The total length of the tibia (distance from The total length of the tibia (distance from the upper edge of the knee joint to the the upper edge of the knee joint to the distal end of the bone) and the femur distal end of the bone) and the femur (distance from the upper edge of the hip (distance from the upper edge of the hip joint to the distal end of the bone) were joint to the distal end of the bone) were measured using an electronic caliper to measured using an electronic caliper to the nearest .1mmthe nearest .1mm

• The coefficient of variance (CV) (mean The coefficient of variance (CV) (mean +/- standard deviation) was calculated +/- standard deviation) was calculated from 10 repeated measurements. from 10 repeated measurements.

• The tibia and femural diaphyses were The tibia and femural diaphyses were scanned at the midpoint because this scanned at the midpoint because this area is almost exclusively cortical area is almost exclusively cortical bone. bone.

• The metaphyses of the tibia and femur The metaphyses of the tibia and femur were scanned at a point located 11.5 were scanned at a point located 11.5 and 16%, respectivley, of total bone and 16%, respectivley, of total bone length from the proximal tip because of length from the proximal tip because of the high content of trabecular bone.the high content of trabecular bone.

AnalysisAnalysis

• They used the total cross-sectional They used the total cross-sectional area (including marrow cavity and area (including marrow cavity and cortical bone, in mmcortical bone, in mm22), cortical area ), cortical area (mm(mm22), cortical thickness (mm), ), cortical thickness (mm), cortical bone mineral content cortical bone mineral content (mg/mm), cortical bone mineral (mg/mm), cortical bone mineral density (BMD= mg/cmdensity (BMD= mg/cm33), total BMD, ), total BMD, and periosteal circumference (mm).and periosteal circumference (mm).

• This was also done for trabercular This was also done for trabercular bone in both the femur and the tibia.bone in both the femur and the tibia.

Statistical AnalysisStatistical Analysis The results obtained were evaluated The results obtained were evaluated

by a one-way analysis of variance by a one-way analysis of variance followed by a post hoc Fisher’s followed by a post hoc Fisher’s protected least difference test. protected least difference test. Differences were considered Differences were considered significant at p < .05. If values are significant at p < .05. If values are below .05 then a difference between below .05 then a difference between groups is real and groups is real and notnot due to chance. due to chance. In this case, In this case, smallersmaller p-values are p-values are better than better than largerlarger ones. ones.

ResultsResults• The tibial diaphyseal total BMD in females The tibial diaphyseal total BMD in females

from Lake Apopka was significantly greater from Lake Apopka was significantly greater than that of females from Lake Woodruff than that of females from Lake Woodruff (table 1).(table 1).

• Tibial trabecular BMD was 34% greater in Tibial trabecular BMD was 34% greater in Lake Apopka (table 2).Lake Apopka (table 2).

• In addition, trabecular bone mineral content In addition, trabecular bone mineral content was 75% greater in Lake Apopka (table 2) was 75% greater in Lake Apopka (table 2) and can be seen in figure 2.and can be seen in figure 2.

• Femural metaphyseal total BMD was 12% Femural metaphyseal total BMD was 12% greater in Lake Apopkagreater in Lake Apopka

• Femural trabecular BMD was 30% greater in Femural trabecular BMD was 30% greater in females from Lake Apopka (table 4).females from Lake Apopka (table 4).

DiscussionDiscussion• Five variables associated with bone Five variables associated with bone

structure differed significantly between structure differed significantly between the two groups of alligators.the two groups of alligators.

• 4 of the 5 were metaphyseal variables.4 of the 5 were metaphyseal variables.• The metaphysis seems to be more The metaphysis seems to be more

sensitive to alteration compared to the sensitive to alteration compared to the diaphysis relative to the exposure to diaphysis relative to the exposure to compounds present in Lake Apopka.compounds present in Lake Apopka.

• Unknown phenomenon has several Unknown phenomenon has several hypotheses.hypotheses.

Hypotheses for abnormal Hypotheses for abnormal growth in metaphysisgrowth in metaphysis

• 1. Metaphyseal reagion might have a higher 1. Metaphyseal reagion might have a higher metabolic rate the the diaphyseal region metabolic rate the the diaphyseal region because of the higher content of trabecular because of the higher content of trabecular bone. bone.

• 2. Hormones influence skeletal morphology 2. Hormones influence skeletal morphology and physiology, but not all parts of the bone and physiology, but not all parts of the bone show similar responses.show similar responses.

• 3. Contaminants can alter hormonal 3. Contaminants can alter hormonal homeostasis would influence the morphology homeostasis would influence the morphology and function of systems dependent on those and function of systems dependent on those horomones.EDC’s can affect different hormonal horomones.EDC’s can affect different hormonal systems such as:systems such as:– Thyroid hormonesThyroid hormones critical for growth, critical for growth,

differentiation, and metabolic regulationdifferentiation, and metabolic regulation– Sex steroidsSex steroids which regulate the reproductive which regulate the reproductive

functions but have effects throughout the body.functions but have effects throughout the body.

Discussion Cont.Discussion Cont.• Results show that alligators from Lake Results show that alligators from Lake

Apopka are exposed to estrogenic Apopka are exposed to estrogenic compounds, because they observed an compounds, because they observed an increase in both total and trabecular BMD, increase in both total and trabecular BMD, a characteristic of estrogen exposure in a characteristic of estrogen exposure in vertebrates.vertebrates.

• Most likely these findings are due to either Most likely these findings are due to either increased plasma levels of 17ß-estranol or increased plasma levels of 17ß-estranol or direct effect of contaminates on processes direct effect of contaminates on processes controlling bone homeostasis.controlling bone homeostasis.

• Another possibility is an effect on both Another possibility is an effect on both osteoblasts and osteoclasts resulting in osteoblasts and osteoclasts resulting in the production and release of growth the production and release of growth factor ß which may mediate actions of factor ß which may mediate actions of estrogen in bone.estrogen in bone.

My discussionMy discussion• This article is important because it This article is important because it

demonstrates that the alterations in demonstrates that the alterations in morphology and physiology induced in free-morphology and physiology induced in free-ranging individuals by environments ranging individuals by environments contaminated with endocrine-active contaminated with endocrine-active compounds are not limited to just a few compounds are not limited to just a few systems, such as the repoductive or systems, such as the repoductive or immune system.immune system.

• I would have included males into the study I would have included males into the study and also see if these differences persist into and also see if these differences persist into adulthood.adulthood.