Chemicals
The aim of the present study was to determine whether developmental DDT exposures that impair thermogenesis later in life alter sympathetic innervation of BAT. To mimic the typical commercial formulation of DDT prior to its ban in the United States, the DDT used to dose animals was a combination of 77.2% p,p’-DDT (98.5% purity neat, AccuStandard, New Haven, CT, USA) and 22.8% o,p’-DDT (100% purity neat, AccuStandard). For the DDE exposure, only the isoform p,p’-DDE (100% purity neat, AccuStandard) was used. Both DDE and the DDT mixture were separately dissolved in the vehicle (Veh), organic extra virgin olive oil from Italian grown olives (Nugget Markets, Woodland, CA, USA), at a concentration of 1.7 mg/kg (DDT) and 1.31 mg/kg (DDE), which was administered daily by oral gavage (10 µL/kg body weight per day). Dosing solutions were prepared at the beginning of the experiment, stored at room temperature (RT, 21–23⁰C) in a gas-tight container, and used within two months. For indirect calorimetry experiments, the β3 adrenergic receptor (AR) agonist CL316,243 (CAS No: 151126-84-0, Cat #1499, 98.1% purity neat; TOCRIS) was dissolved daily in sterile phosphate-buffered saline (PBS, pH 7.4; cat#59321C, Sigma, St. Louis, MO, USA) at a concentration of 0.1 mg/mL and warmed to 37⁰C prior to intraperitoneal (i.p.) injection at a volume of 10 µL/g body weight.
Certified reference standards for HPLC quantification were purchased from Accustandard, including o,p’ DDT, p,p’-DDT, o,p’-DDE, p,p’-DDE, 13C12 labeled p,p’-DDE, D8 labeled p,p’-DDT, phenanthrene D-10, and chrysene D-12. Acetone (99.8%, HPLC grade), n-hexane (≥ 99%), and dichloromethane (99.8%, HPLC grade) were purchased from Fisher Scientific.
Animal handling and exposures
Animals were maintained in facilities fully accredited by AAALAC International, and all studies were performed with regard for alleviation of pain and suffering under protocols approved by the UC Davis Institutional Animal Care and Use Committee (IACUC protocol #18938). All animal experiments were conducted in accordance with the ARRIVE guidelines and the National Institutes of Health guide for the care and use of laboratory animals (NIH publication No. 8023, revised 1978). Male and female 8-week-old C57BL/6J mice were obtained from Jackson Laboratories (Sacramento, CA, USA). Except during mating, adult male and female mice were separately housed with 2–5 animals per cage in standard plastic cages under controlled environmental conditions (12 h light/dark cycle, 40–50% humidity). Litters were kept with the dam until weaned at postnatal day (PND) 21. Room temperature was maintained between 21–23⁰C, which is considered a minor cold stress with the potential for stimulating low-grade sympathetic activation (30, 31). All animals had access to food (5053 Purina diet) and water ab libitum. Upon receipt from the vendor (Jackson Laboratories), 8-week-old male and nulliparous female C57BL/6J mice were acclimated for one week prior to timed mating. Pregnancy was determined by the presence of a postcoital vaginal plug. Primigravid females were randomly assigned to experimental groups using a random number generator, using a priori decisions to maximize litter number per group based on sex per litter; thus, final counts for sex-based longitudinal rectal temperature were n = 14 (Veh), 15 (DDT) or 7 (DDE) non-littermates/group for a total of 36 litters, and all remaining analyses in females at n = 7 non-littermates/group for a total of 21 litters, unless otherwise stated in the figure or table legends. Once assigned to an experimental group, dams were orally gavaged (10 µL/g body weight) with DDT, DDE, or Veh from gestational day (GD) 11.5 until postnatal day (PND) 5. This period encompasses crucial ontological events for autonomic ganglionic formation, establishment of peripheral sympathetic synaptic connections, sympathetic target tissue innervation, and BAT activation by the sympathetic nervous system (32–34). At PND 5, litters were culled to 6 random pups to normalize lactational pesticide transfer, and pups were weaned at PND 21. At 4 months of age, female offspring were euthanized by exsanguination under isoflurane anesthesia.
Temperature measurements and indirect calorimetry
Longitudinal core body temperature was measured in female offspring in four-week intervals beginning at 5 weeks after birth using a thermocoupled probe (RET-4, Physitemp, Clifton, NJ, USA) inserted into the rectum to a depth of 5 mm.
Thermoneutral zone (TNZ) was assessed using indirect calorimetry with implanted temperature recorders. DST nano-T temperature recorders (Star-Oddi, Gardabaer, Iceland) were implanted intraperitoneally under anesthesia (2–4% isoflurane). For analgesia, animals received meloxicam (2–10 mg/kg, subcutaneous, SC) pre-operatively and, as needed, buprenorphine (0.05–0.1 mg/kg, SC) post-operatively. Mice were moved to the calorimetry room at least ten days after surgery. Mice were acclimated to the calorimetry room for 24 h while housed in acclimation chambers. The morning of TNZ analysis, food was removed from the acclimation chamber to ensure that calorimetry readings were not influenced by foraging activity. To evaluate the TNZ, a mouse in an acclimation chamber was transferred to a calorimetry chamber within a temperature-controlled cabinet set at 12° C. Cage temperature was measured using ambient temperature sensors (DS1922L-F5#, iButtonLink, LLC) attached to the inside of the metabolic cage lid. Previous tests measuring temperature within the calorimeter chambers were used to select the chamber location for the studies that best matched the target temperatures. Energy expenditure was determined by indirect respiration calorimetry (35) recorded every 5 sec in the Comprehensive Lab Animal Monitoring Systems (CLAMS, Columbus Instruments) unit at 12 °C for 60 min, at 18 °C, 24 °C, 28 °C, and 30 °C for 45 min each, and at 34 °C and 36 °C for 30 min each. The body temperature recorders were programmed to record every 5 min from the beginning of the 12 °C interval until the beginning of the 30 °C calorimetry measurements and for every minute for the remainder of the calorimetry measurements (30 °C through 36 °C).
Oxygen consumption and heat production in response to CL316,243 was determined by indirect calorimetry (35). Four-month old female mice were individually housed in CLAMS metabolic chambers and acclimated for at least three days prior to data acquisition. On the day of pharmaceutical intervention, mice had food and water removed for 4 h prior to intraperitoneal injection (IP) injection with either CL316,243 (0.1 mg/kg) or Veh (sterile phosphate buffered saline [PBS], pH 7.4; #59321C, Sigma-Aldrich, St. Louis, Missouri, USA) by researchers blinded to experimental groups. Subsequent indirect calorimetry metrics were recorded for the next 60 min, with sampling frequency at one min intervals. Measurements were averaged over a 20 min window after chamber gas equilibration, from post-injection minute 11 to 31 to encompass a period of peak BAT activation by CL316,243 (36). Room temperature was maintained between 21–23⁰C, rather than at TNZ due to the lack of a DDT-related shift of TNZ (Table S1) and kept on a 12 h light/dark cycle.
Histology and immunofluorescence
BAT was collected from the intrascapular region immediately following euthanasia. BAT was fixed in 4% (w/v) paraformaldehyde (PFA; Sigma) diluted in 0.2M phosphate buffer (0.2M Na2HPO4, 0.2M NaH2PO4, pH 7.2) for 24 h, then transferred to 30% (w/v) sucrose (Sigma) diluted in phosphate buffered saline (PBS; 10 mM Na2HPO4, 1.76 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl; pH 7.4; Sigma) for long-term storage at 4⁰C. Fixed BAT samples were transferred into 70% v/v ethanol in water, then embedded in paraffin. Three 5 µm thick sections were obtained at 100 µm intervals per tissue sample and stained with hematoxylin and eosin (H&E). H&E-stained sections were analyzed by the UC Davis Comparative Pathology Laboratory (UC Davis School of Veterinary Medicine). A pathologist blinded to study groups examined BAT sections for inflammation, degeneration and necrosis. Vacuolation of BAT adipocytes was subjectively classified as the ratio of cells with small to large intracytoplasmic vacuoles. This quantification avoided obvious tissue edge and blood vessels, and assumed unstained round cellular droplets to be lipids contained within intracytoplasmic vacuoles.
For immunohistochemical analyses, PFA-fixed BAT was embedded in optimal cutting temperature (OCT) compound (#4583, Tissue-Tek, Sakura Finetek, Torrance, CA, USA) and flash-frozen. Three sections per sample were serially sliced 10 µm thick mounted on Superfrost Plus slides (Cat#12-550-15, Fisher Scientific) using a cryostat set to -30⁰C (Microm HM550 cryostat, Thermo Scientific), and slides were stored at -80⁰C until further processed. Once slides were brought to room temperature (RT), heat-mediated antigen retrieval was performed on slides submerged in citrate buffer (10 mM sodium citrate in 0.05% (v/v) Tween-20 [Sigma-Aldrich] diluted with MilliQ H2O, pH 6.0) using a vegetable steamer for 30 min to attain a minimum temperature of 60⁰C. Following antigen retrieval, samples were blocked for 1 h at RT in blocking buffer, which was 10% (v/v) normal goat serum (Vector Laboratories, Burlingame, CA, USA), 2% (w/v) bovine serum albumin (Sigma), 0.05% (v/v) Tween-20 in PBS (pH 7.4, Sigma). Sections were then immunoreacted for 48 h at 4⁰C with the following primary antibody cocktail diluted in blocking buffer: mouse anti-neuropeptide Y (NPY) (1:250, monoclonal IgG1, ab112473, RRID:AB_10861167; Abcam, Cambridge, MA, USA) and rabbit anti-tyrosine hydroxylase (TH) (1:1000, polyclonal, Cat #AB152, RRID: AB_390204; EMD Millipore, Temecula, CA, USA). After incubation with primary antibodies, sections were washed with PBS containing 0.05% (v/v) Tween-20 and then incubated for 1 hr at RT with AlexaFluor-488 goat anti-mouse IgG F(ab’)2 (lot#1812170, Invitrogen, Waltham, MA, USA) and AlexaFluor-568 goat anti-rabbit IgG (lot#1871167, Invitrogen) each diluted to 1:1000 in PBS (pH 7.4) containing 0.05% (v/v) Tween-20. Sections were then washed and coverslipped with ProLong Gold Antifade Mountant with 4′,6-diamidino-2-phenylindole, (DAPI, P36931, ThermoScientific) and left to cure overnight before imaging.
To immunostain stellate ganglia, unfixed stellate ganglia collected immediately upon euthanasia were immediately embedded in OCT (Tissue-Tek) and flash frozen. Samples were serially cryosectioned at 10 µm, permeabilized for 5 min in 0.2% (v/v) Triton X-100, and then blocked for 2 h at RT with blocking buffer. Slides were then immunoreacted overnight at 4⁰C with primary antibody cocktail diluted in blocking buffer: rabbit anti-chapsyn-100 (PSD93) (1:500, polyclonal, AB5168-200UL, RRID:AB_91716; EMD Millipore) and mouse anti-bassoon (1:500, ADI-VAM-PS003-F, RRID:AB_11181058; ENZO Life Sciences, Plymouth Meeting, PA, USA). After incubation with primary antibodies, sections were washed with PBS containing 0.02% (v/v) Tween-20 and then incubated for 1 hr at RT with AlexaFluor-488 goat anti-mouse IgG F(ab’)2 (lot#1812170, Invitrogen) and AlexaFluor-568 goat anti-rabbit IgG (lot#1871167, Invitrogen) each diluted to 1:1000 in PBS with 0.02% (v/v) Tween-20. Sections were then washed and coverslipped with ProLong Gold Antifade Mountant with DAPI (P36931, Thermo Scientific) and left to cure overnight before imaging. Staining batches were evenly stratified across all treatment groups to minimize batch effects, and appropriate controls were included in each stain batch for downstream inter-batch comparisons and thresholding determinations to optimize signal:noise during quantification.
Catecholamine concentrations
BAT (n = 7/group) was collected immediately after euthanasia and flash frozen. Tissue was homogenized in perchloric acid (300 µl, 0.1 M) containing dihydroxybenzylamine (1.0 µM) internal standard. After homogenization, all samples were centrifuged (13000 g for 5 min). Catecholamines were purified from an aliquot (100 µL) of the supernatant by alumina adsorption. Norepinephrine (NE) and its metabolite dihydroxyphenylglycol (DHPG) were measured by HPLC with electrochemical detection as described previously (37–39). Detection limits were ~ 0.05 pmol with recoveries from the alumina extraction > 60%.
DDT metabolite concentration quantification
Tissue concentrations of o,p’-DDT, p,p’-DDT, o,p’-DDE, and p,p’-DDE were measured in BAT from 4-month-old mice using gas chromatography (GC)-MS following methods described previously (2). Briefly, 30 mg flash frozen BAT samples (n = 3/group) were extracted using a QuEChERS-based extraction with 1:1:1 hexane:acetone:dichloromethane. Briefly, the samples were spiked with 10 µL of a solution containing 13C12 labeled p,p’-DDE and D8 labeled p,p’-DDT as internal standards to assess recovery. The samples and 5 mL of the solvent mixture were added to 7-dram amber vials and vortexed mixed. The supernatant was added to a 15 mL QuEChERS tube containing 150 mg dispersive C18 powder and 900 mg anhydrous MgSO4 (United Chemical Technologies, Bristol, PA). The Quechers tube was shaken for 15 minutes on a rotary shaker (Fisherbrand) and centrifuged (5 minutes). The supernatant was transfer to a glass centrifuge tube. These steps were repeated two more times with 3 mL of 1:1:1 hexane:acetone:dichloromethane (11 mL total). The final extract was evaporated down to 150 µL under nitrogen (Organomation 30 position Multivap Nitrogen Evaporator). After extraction, samples were spiked with phenanthrene-D10 and chrysene D-12 as internal standards to ensure injection consistency during GC-MS analysis.
Sample extracts were then analyzed on a high-resolution GC Q-Exactive Orbitrap MS (Thermo Scientific) equipped with a Thermo Trace 1300 gas chromatograph and TriPlus RSH Autosampler. The extracts (3 µL) were injected into a 290oC split/splitless inlet operated in split-less mode. The analytes were separated on a Restek Rxi-35Sil MS column (30 m x 0.25 mm inner diameter x 0.25 µm film thickness) with Helium (99.999% purity) as the carrier gas (1 mL/min). The oven temperature ramp began at 130oC for 0.5 min, increased 30oC/min to 235oC and held for 4 min, 10oC/min to 275oC, and 50oC/min to 320oC and held for 10 min, with a total run time of 23 min. The transfer line was maintained at 300oC and the EI source temperature at 250 oC. The MS was operated in full scan mode, with a scan range of 150 to 350 m/z. The most abundant peak in the mass spectrum was used to quantify each analyte and identify was confirmed using the ratio of two confirming ions and retention times (Table 1). Quantification was performed using a ten-point calibration curve prepared by serial dilution of calibration standards in hexane (.007 to 30 µg L− 1). The limited of detection (LOD) for each target analyte was determined from seven injections of calibration standards and calculated as previously described (40, 41) using the following equation:
where s is the standard deviation of the 7 injections, t is the student’s t-value, df is the degrees freedom, and α is the level of significance (for n = 7 and 𝛼 = 0.01, t = 3.14). Limits of detection (LOD) per metabolite in a 30 mg sample were as follows: 0.026 pg/mg BAT (p,p’-DDT), 0.269 pg/mg BAT (p,p’-DDE), 0.153 pg/mg BAT (o,p’-DDT), and 0.104 pg/mg BAT (o,p’-DDE).
Table 1
Retention time and ions monitored to quantify and confirm DDE metabolites.
Analyte
|
RT (min)
|
Quantifying Ion
(m/z)
|
Confirming Ion 1
(m/z)
|
Confirming Ion 2
(m/z)
|
o,p'-DDE
|
8.36
|
245.9999
|
247.9968
|
317.9345
|
o,p'-DDT
|
10.64
|
235.0076
|
165.0699
|
237.0047
|
p,p'-DDE
|
9.10
|
245.9999
|
247.9968
|
317.9345
|
p,p'-DDT
|
11.35
|
235.0076
|
165.0699
|
237.0047
|
4,4'-DDE (13C12)
|
9.17
|
260.0370
|
188.1021
|
258.0400
|
4,4'-DDT (D8)
|
11.27
|
243.0576
|
173.1200
|
245.0549
|
RT = retention time |
Data processing and analyses
The lower critical temperature (LCT) of the TNZ, previously reported to be between 26–32 °C for varying strains of laboratory mice (42, 43), was calculated using a segmental linear model of energy expenditure versus calorimeter chamber temperature with the slope 2 = 0. Although the upper critical temperature (UCT) of the TNZ is frequently defined as the ambient temperature where heat stress induces an increase in energy expenditure (44), we did not consistently observe increases in energy expenditure even at ambient temperatures where body temperature was increased (See Supplementary Table 1, Additional file 1). Therefore, we followed the approach suggested by Abreu-Vieira et al. (45) and used an increase in body temperature to identify the UCT. The UCT was calculated using a segmental linear model of core body temperature versus calorimetry chamber temperature with the slope 1 = 0. Energy expenditure in mice was measured at temperatures ranging from 12 to 36 °C, after which Scholander plots were constructed (46) to determine the effect of ambient temperature on energy expenditure and identify the TNZ (Table S1). Only body temperature data collected at calorimetry chamber temperatures above 28° C were used for the calculations to avoid fluctuations in body temperature that were more common at colder temperatures.
Images of immunofluorescence were captured using an Olympus IX81 wide-field microscope with either a 20x SAPO BF or 60x SAPO BF objective lens for BAT and stellate, respectively, with consistent exposure times across all samples. Regions of interest (ROIs) were randomly selected per tissue section (n = 1–3 ROI technical replicates within each section, depending on the availability of non-overlapping viewing fields and tissue size) for each of the three tissue sections per slide, for a total of n = 3–9 ROIs per sample evaluated. Automated, nonbiased colocalization analyses were performed using MetaXpress Image Analysis Software (MetaXpress Image Acquisition and Analysis Software v6.1, Molecular Devices Corp., USA) based on a priori-designated signal:noise ratios per channel and background fluorescence thresholds for tissue-specific immunopositive staining. Target-specific controls lacking primary or secondary antibodies were included in each staining batch to determine thresholds for immunopositive fluorescence and adjust for batch effects.
All data were presented as the mean ± standard error of the mean (SE). Data were assessed for normality using Kolmogorov-Smirnov or Shapiro-Wilk normality tests, and outliers were removed based on identification via Grubb’s test (GraphPad Prism version 8.4.0, GraphPad Software Inc., San Diego, CA, USA). Histological analyses and catecholamine measurements were then assessed using either nonparametric Kruskal-Wallis tests with Dunn’s correction for data not normally distributed (e.g. H&E histology data) or analysis of variance (ANOVA) with Tukey’s correction for normally distributed data (e.g. immunofluorescence and catecholamine outcomes) using GraphPad PRISM 7. Body temperature was normally distributed and thus least square means differences between categorical effect of perinatal treatment were analyzed at each age using general linear models (PROC GLM, SAS v9.4, Statistical Analysis System (SAS) Institute, Cary, NC, USA). Indirect calorimetry-derived parameters were modeled with the fixed effects of perinatal exposure, acute (agonist or Veh) exposure, and their interaction, while accounting for the repeated measures from individual mice and resulting covariance structure in a mixed linear model that allows the data to exhibit within- individual correlation (PROC MIXED, SAS v9.4). All parameters were statistically analyzed at a significance threshold of p < 0.05 for main effects and p < 0.1 for interaction effects.