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Published in final edited form as: Bone. 2024 Apr 26;185:117111. doi: 10.1016/j.bone.2024.117111

Six Months of Voluntary Alcohol Consumption in Male Cynomolgus Macaques Reduces Intracortical Bone Porosity Without Altering Mineralization or Mechanical Properties

Amida H Kuah 1, Lara H Sattgast 1, Kathleen A Grant 2, Steven W Gonzales 2, Rupak Khadka 2, John G Damrath 3, Matthew R Allen 4,5, David B Burr 4,5, Joseph M Wallace 5, Gianni F Maddalozzo 1, Mary Lauren Benton 6, Laura M Beaver 1,7, Adam J Branscum 8, Russell T Turner 1,9, Urszula T Iwaniec 1,9,*
PMCID: PMC466935  NIHMSID: NIHMS1993670  PMID: 38679220

Abstract

Chronic heavy alcohol consumption is a risk factor for low trauma bone fracture. Using a non-human primate model of voluntary alcohol consumption, we investigated the effects of 6 months of ethanol intake on cortical bone in cynomolgus macaques (Macaca fascicularis). Young adult (6.4 ± 0.1 years old, mean ± SE) male cynomolgus macaques (n = 17) were subjected to a 4-month graded ethanol induction period, followed by voluntary self-administration of water or ethanol (4% w/v) for 22 h/d, 7 d/wk for 6 months. Control animals (n = 6) consumed an isocaloric maltose-dextrin solution. Tibial response was evaluated using densitometry, microcomputed tomography, histomorphometry, biomechanical testing, and Raman spectroscopy. Global bone response was evaluated using biochemical markers of bone turnover. Monkeys in the ethanol group consumed an average of 2.3 ± 0.2 g/kg/d ethanol resulting in a blood ethanol concentration of 90 ± 12 mg/dl in longitudinal samples taken 7 hours after the daily session began. Ethanol consumption had no effect on tibia length, mass, density, mechanical properties, or mineralization (p > 0.642). However, compared to controls, ethanol intake resulted in a dose-dependent reduction in intracortical bone porosity (Spearman rank correlation = −0.770; p < 0.0001) and compared to baseline, a strong tendency (p = 0.058) for lower plasma CTX, a biochemical marker of global bone resorption. These findings are important because suppressed cortical bone remodeling can result in a decrease in bone quality. In conclusion, intracortical bone porosity was reduced to subnormal values 6 months following initiation of voluntary ethanol consumption but other measures of tibia architecture, mineralization, or mechanics were not altered.

Keywords: Bone microarchitecture, biomechanics, Raman spectroscopy, ethanol, non-human primate

1. Introduction

Chronic alcohol abuse is a risk factor for low trauma fractures (Kanis et al., 2005). Due to its importance in structural support, alcohol-induced modifications in cortical bone may contribute to the alcohol-associated increases in fracture risk (Clarke, 2008). Although the precise mechanisms are uncertain, the detrimental effects of alcohol on bone are mediated, in part, by disturbances in bone remodeling. Bone is remodeled both over time (random remodeling) and in response to fatigue microdamage resulting from normal everyday activity (targeted remodeling) (Eriksen, 2010). Bone remodeling depends on the coordinated actions of bone resorbing osteoclasts and bone forming osteoblasts. A long-term imbalance in the rate of formation and the rate of resorption will act to alter the quantity, quality, and the structural integrity of bone (Bolamperti et al., 2022). Bone biopsies (usually iliac crest) are used to ascertain bone remodeling dynamics in humans, but procedures for procuring biopsies are invasive. Consequently, tissue level studies evaluating the effects of alcohol consumption on bone remodeling in humans are uncommon and the few investigations performed have focused on cancellous bone (Gaddini et al., 2016).

Due to the considerable challenges in performing alcohol studies in humans, our understanding of the effects of alcohol on the skeleton is based on animal models, primarily rodents (Gaddini et al., 2016). Rodent studies have focused on bone during growth; only a few have evaluated bone remodeling in adults (Wahl et al., 2006, Hogan et al., 2001, Turner et al., 2001b, Sibonga et al., 2007). In addition, small rodents do not naturally exhibit intracortical bone remodeling, an important contributor to bone turnover. In contrast, physiological intracortical bone remodeling similar to humans is observed in non-human primates. In previous studies, male rhesus macaques given free access to alcohol for 12 months had significantly reduced initiation of intracortical bone resorption, resulting in a reduction in cortical bone porosity (Gaddini et al., 2015). This may be important because disruption of bone remodeling by chronic consumption of alcohol has potential to lead to degradation of bone quality by preventing repair of fatigue microdamage and/or by permitting tissue-level changes in bone mineralization and collagen crosslinking (Boivin and Meunier, 2003, Burr et al., 1997, Seref-Ferlengez et al., 2015). Twenty months of heavy alcohol consumption had only a minor influence on the composition and material properties of cortical bone in young adult male rhesus macaques (Shin et al., 2024). However, in the alcohol group elastic modulus was increased and crack growth toughness was decreased, changes consist with degradation of bone quality.

Taken together, evidence supports the hypothesis that suppressed intracortical bone remodeling contributes to an alcohol-mediated reduction in bone quality. However, findings to date are limited to one primate species and have not adequately addressed whether alcohol influences bone composition or mineralization of bone matrix. In the current study, we address gaps in prior research by evaluating a second species of non-human primates (cynomolgus macaques) subjected to a shorter interval (6 months) of “open access” (22 h/d) to ethanol. As before, we evaluated the impact of chronic ethanol consumption on bone mass, density, and cortical architecture. In addition, we evaluated the impact of ethanol on bone matrix composition and mineralization using Raman spectroscopy and on mechanical properties by strength testing.

2. Materials and Methods

2.1. Animals

The study population consisted of 23 (n = 6 control and 17 ethanol) young adult (6.4 ± 0.1 years old at treatment initiation, mean ± SE) male cynomolgus macaques (Macaca fascicularis) representing 2 consecutive cohorts subjected to the same experimental protocol (Figure 1) and well-matched for age and body weight: cohort 9 (n = 3 control and 8 ethanol) and cohort 13 (n = 3 control and 9 ethanol) (Walter et al., 2020). The animals were obtained from SNBL USA (Everett, WA) and were of Cambodian origin. The monkeys were individually housed in standard primate caging (0.8 × 0.8 × 0.9 m) in a humidity-controlled (65%), temperature-controlled (20–22 °C), and light-controlled (light on 0700–1800 hours) room that allowed visual, auditory, and olfactory contact among the animals. Ethanol intake was recorded continuously and blood ethanol concentration (BEC) was measured every 5 days. Details of the daily intakes and BECs of all subjects are available through the Monkey Alcohol Tissue Research Resource (MATRR.com; please refer to cohorts 9 and 13) (Daunais et al., 2014). All of the animals were maintained in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the experimental protocol was approved by the Institutional Animal Care and Use Committee at the Oregon Health & Science University where the studies were conducted.

Figure 1.

Figure 1.

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Experimental protocol: the monkeys were trained to use an operant panel and induced to drink increasing volumes of an ethanol solution in a step-wise fashion (0.0 g/kg, 0.5 g/kg, 1.0 g/kg, and 1.5 g ethanol/kg body weight over 4 consecutive 30-day periods. The monkeys started with water only during the initial period. This was followed by a 6-month voluntary drinking phase where monkeys in the ethanol group were given free access to water and ethanol (4% w/v in water) for 22 h/d, 7 d/wk.

2.2. Experimental Protocol

The experimental protocol is described in detail elsewhere (Grant et al., 2008, Walter et al., 2020). In brief, the protocol consisted of two phases, an induction phase (4 months) and a voluntary drinking phase (6 months). During induction, the monkeys were trained to use an operant panel and induced to drink increasing volumes of an ethanol solution in a step-wise fashion over 4 consecutive 30-day periods (Figure 1). The monkeys started with water only during the initial period. This was followed by incremental increases in ethanol starting with 0.5 g/kg body weight, followed by 1.0 g/kg body weight and subsequently by 1.5 g/kg body weight. Body weights were recorded weekly. During the 6-month voluntary drinking phase, monkeys in the ethanol group were given free access to water and ethanol (4% w/v in water) for 22 h/d, 7 d/wk. Control animals self-administered, and consumed, a volume of maltose-dextrin solution isocalorically equivalent to the previous week’s average volume of ethanol consumed by an ethanol animal assigned at baseline (yoked control).

Venous blood was collected prior to ethanol induction (baseline) in awake monkeys (the animals were trained to present their legs in their home cage for blood collection) and at necropsy for measurement of markers of bone turnover, cortisol, and dehydroepiandrosterone sulfate (DHEAS) and plasma samples (500 μl) were stored at −80°C until analysis was performed. For tissue collection, subjects were sedated with ketamine (10mg/kg, intramuscular) and euthanized with pentobarbital (30mg/kg, intravenous). Right tibiae were harvested, placed in 70% ethanol, and stored at 4°C until analysis [dual-energy X-ray absorptiometry (DXA), microcomputed tomography (μCT), and histomorphometry]. Left tibiae were wrapped in saline-soaked gauze and stored frozen at −20°C until analysis (biomechanics and Raman spectroscopy). Freeze thaw cycles were kept to a minimum and all specimens were treated similarly (Turner and Burr, 1993).

2.3. Blood Ethanol Concentrations

Blood samples (20 μl) were collected from the medial saphenous vein in awake monkeys approximately every 5 days at 7 hours into the daily drinking session. Samples were diluted in 500 μl sterile water, placed in airtight containers, and stored at −4°C until assayed using headspace gas chromatography (Agilent Technologies, Santa Clara, CA).

2.4. Markers of Bone Turnover, Cortisol, and DHEAS

Plasma carboxyterminal cross-linking telopeptide of type 1 collagen (CTX) and osteocalcin were determined at baseline and necropsy. CTX and osteocalcin are markers of global bone resorption and formation, respectively. The Endocrine Technologies Core (ETC) at the Oregon National Primate Research Center conducted the assays using a Roche Cobas e411 Automated Clinical Platform (Roche Diagnostics, Indianapolis, IN). The ETC validated the assays for use in nonhuman primates. The assay ranges of the CTX and osteocalcin assays were 0.01 – 6.00 ng/ml and 0.5 – 300 ng/ml, respectively. Intra-assay coefficient of variation (CV) for CTX was 1.1% and intra-assay CV for osteocalcin was 7.8%. No inter-assay CV was calculated for these specimens as all samples were measured on the same day. Plasma cortisol and DHEAS were assayed as described (Allen et al., 2018), also by the ETC.

2.5. Dual-Energy X-Ray Absorptiometry

Bone area (cm2), bone mineral content (BMC, g), and areal BMD (g/cm2) were determined in the right tibiae using DXA (Hologic Discovery A, Waltham, MA; Hologic APEX System Software, Version 3.1.1). Quality control check was performed against the Anthropomorphic Spine Phantom and Small Animal Step Phantom provided by the manufacturer.

2.6. Microcomputed Tomography

μCT was used for nondestructive 3-dimensional evaluation of cortical bone architecture. Right tibial length was measured as the distance between the proximal tip of the intercondylar eminence and the distal tip of the medial malleolus. The distal third of the tibia was then excised using an IsoMet® Low Speed Saw (Buehler, Lake Bluff, IL) and scanned in 70% ethanol at a voxel size of 30 × 30 × 30 μm (55 kVp, 145 μA, and 200 ms, 500 projections/rotation) on a Scanco μCT40 scanner (Scanco Medical AG, Basserdorf, Switzerland). Evaluations were conducted with filtering parameters sigma and support set to 0.8 and 1, respectively. Thirty-three consecutive slices (1.0 mm) of cortical bone (at proximal end of the distal third of the tibia) were analyzed at a threshold of 245 (gray scale of 0–1000) determined empirically. This threshold corresponds to 374 mg hydroxyapatite/cm3. Cortical measurements included (1) total tissue volume (cortical and marrow volume, mm3), (2) cortical volume (mm3), (3) marrow volume (mm3), (4) cortical thickness (μm), and (5) polar moment of inertia (an estimate of bone strength in torsion, mm4).

2.7. Bone Histomorphometry

A 50-μm thick cross-section of bone was removed from the proximal end of the scanned portion of the tibia using the IsoMet® Low Speed Saw. The cross-sections were ground on a roughened glass surface (using 220-grit aluminum oxide powder) to an approximate thickness of 25 μm for histomorphometric evaluation. Cortical porosity was determined by measuring the number and cross-sectional area of Haversian canals in the entire tibial cross-section. Pores smaller than 0.01 mm2, which included osteocyte lacunae, were not measured. Canal number and area were normalized to bone area (number/mm2 and %, respectively). All histomorphometric data were collected using an Olympus BH2 Microscope (Olympus, Shinjuku, Tokyo, Japan) equipped with an Olympus DP71 microscope digital camera (Olympus, Shinjuku, Tokyo, Japan) and attached to a computer system with OsteoMeasure software (OsteoMetrics, Atlanta, GA; version 3.3.0.2).

2.8. Mechanical Properties

Left tibiae were tested to failure in a 3-point bending configuration with a support span set at 16 cm (MTS MiniBionix 858). Displacement was applied monotonically in the medial-lateral direction at a loading rate of 0.025 mm/s (medial surface in tension). Bones were kept hydrated with PBS throughout the test. Force and displacement were recorded during the test, and post-testing analysis was performed in MATLAB (MathWorks, Natick, MA) to construct a force-displacement curve. Using standard engineering bending equations, μCT data were then used to calculate stress and strain. The yield point was located using the 0.2% strain offset method on the stress-strain curve, and was then mapped back to the force-displacement curve.

2.9. Raman Spectroscopy

A transverse section of each left tibia was cut 50 mm proximal to the loading region following mechanical testing. Sections were sanded to create a flat surface and polished with a 3 μm diamond suspension. Polished samples were kept frozen in PBS-soaked gauze prior to Raman spectroscopy. Raman spectra were obtained using an InVia Raman Spectrometer (Renishaw, Wotton-under-Edge, United Kingdom). A 785 nm laser was focused on the bone surface through a 50x objective to a spot size of ~1 μm. Spectra were obtained midway between the periosteal and endosteal surfaces in 45° increments around the surface of the bone, resulting in 8 locations per sample. 12 spectra, each with a 12s exposure time, were acquired at each location and averaged to yield a single spectrum per location for analysis. Baseline correction was accomplished using Renishaw WiRE software intelligent fitting. Gaussian peaks were fit to the v1-PO43− and v1-CO32− peaks by second derivative spectroscopy in GRAMS/AI (Thermo Fisher Scientific, Waltham, MA). The extent of matrix mineralization was determined by the mineral/matrix ratio, calculated as the integrated area ratio v1-PO43−/Amide I as previously described (Hammond et al., 2014). Mineral maturity/crystallinity was calculated as the inverse of the full width at half height of the v1-PO43− peak. Type B carbonate substitution was calculated as the area ratio v1-CO32−/v1-PO43− of the fitted Gaussian peaks. Each Raman parameter was calculated for all 8 sites and averaged, yielding a single value for each parameter per sample.

2.10. Statistical Analysis

Mean outcomes for ethanol consuming and control macaques were compared using Welch’s two-sample t-test or the distribution-free Wilcoxon-Mann-Whitney tests (when normality was violated), approaches that do not require equal sample size. A paired t-test or the Wilcoxon signed rank test was used to compare baseline and necropsy mean values of CTX, osteocalcin, DHEAS, and cortisol separately for each group. The differences in mean osteocalcin at baseline and necropsy for the control and ethanol consuming groups were compared using a two-sample t-test, while the corresponding differences for CTX were compared using a Wilcoxon-Mann-Whitney test. Data on percent change from baseline to necropsy were analyzed using one-sample t-tests. Residual analysis and Levene’s test were used to assess normality and homogeneity of variance. Spearman rank correlation was used to determine the relationship between level of alcohol consumption and cortical bone porosity. The Benjamini and Hochberg method for maintaining the false discovery rate at 5% (Benjamini and Hochberg, 1995) was used to adjust for multiple comparisons. Differences were considered significant at p ≤ 0.05. Data are presented as mean ± SE. Data analysis was performed using R version 4.1.2.

3. Results

Characteristics of the study population are presented in Table 1. The control and ethanol groups were well-matched for age and body weight. Monkeys in the ethanol group consumed on average 2.3 ± 0.2 g/kg/d of ethanol which led to a BEC of 90.3 ± 12.3 mg/dl 7 hours after the session onset.

Table 1.

Study population.
Control Ethanol
Age at treatment initiation (years) 6.7 ± 0.05 6.3 ± 0.1
Terminal body weight (kg) 7.9 ± 0.2 8.0 ± 0.2
Average daily ethanol intake (g/kg) 2.3 ± 0.2
Average daily blood ethanol concentration (mg/dl) 90.3 ± 12.3
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Data are mean ± SE, n = 6 control and 17 ethanol-consuming monkeys

The effects of ethanol on intracortical porosity in the distal tibia diaphysis are shown in Figure 2. Canal density (Figure 2A) and size (Figure 2B) did not differ between control and ethanol-consuming monkeys. However, ethanol consumption resulted in lower total intracortical porosity (canal area/bone area; 2.0 ± 0.1% versus 1.2 ± 0.04%, p <0.001) (Figure 2C). There was a strong inverse association between porosity and ethanol intake (Spearman rank correlation = −0.770; p < 0.0001) (Figure 2D). The inverse association continued to be significant (−0.509; p = 0.037) even when control monkeys were excluded.

Figure 2.

Figure 2.

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Effects of 6 months of voluntary ethanol consumption on canal density (A), canal size (B), total intracortical porosity (canal area/bone area) (C), and intracortical porosity in relation of ethanol intake (D) in male cynomolgus macaques. Data are mean ± SE, with dots representing individual subjects; n = 6 for control and n = 17 for ethanol. A Welch’s two-sample t-test was used for assessment of canal density and size and a T-test was used for assessment of intracortical porosity.

The effects of ethanol on plasma markers of bone turnover are shown in Figure 3. CTX, a marker of global bone resorption, did not differ between control and ethanol monkeys at necropsy. However, unadjusted CTX levels in ethanol-consuming monkeys decreased (P=0.004) compared to baseline values (Figure 3A) but following FDR adjustment for multiple comparisons the change was reduced to a trend (p = 0.058). In contrast, there were no changes over time in the control animals. No significant differences with ethanol intake or time were detected in levels of osteocalcin, a global marker of bone formation (Figure 3B). However, there was a trend (P=0.087) for osteocalcin to be lower in ethanol consuming monkeys than controls at necropsy.

Figure 3.

Figure 3.

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Effects of 6 months of voluntary ethanol consumption on plasma carboxyterminal cross-linking telopeptide of type 1 collagen (CTX) (A) and osteocalcin (B) at baseline (pre-ethanol treatment) and necropsy in male cynomolgus macaques. Individual values as well as summary data (mean ± SE, below x-axis legend) are shown. N = 6 for control and n = 17 for ethanol. A paired t-test was used to separately compare baseline and necropsy mean values of CTX (Control group) and osteocalcin (Control and Ethanol groups) while a Wilcoxon signed rank test was used for CTX in the Ethanol group.

The effects of ethanol on plasma levels of DHEAS and cortisol are shown in Figure 4. Neither DHEAS (Figure 4A) nor cortisol levels (Figure 4B) differed with treatment at necropsy: unadjusted, DHEAS values were higher (P <0.04) in ethanol-consuming monkeys compared to controls, but the difference was no longer significant following adjustment for multiple comparisons. DHEAS levels decreased with time (baseline to necropsy) in control but not in ethanol-consuming monkeys. In contrast, cortisol levels decreased with time in both control and ethanol-consuming animals.

Figure 4.

Figure 4.

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Effects of 6 months of voluntary ethanol consumption on plasma dehydroepiandrosterone sulfate (DHEAS) (A) and cortisol (B) at baseline (pre-ethanol treatment) and necropsy in male cynomolgus macaques. Individual values as well as summary data (mean ± SE, below x-axis legend) are shown. N = 6 for control and n = 17 for ethanol. bDifferent from Baseline, P ≤ 0.05. A paired t-test was used to compare baseline and necropsy mean values of DHEAS and cortisol separately for each group.

The effects of ethanol on tibia length, BMC and BMD, and on distal tibia cortical architecture (total volume, cortical volume, marrow volume, cortical thickness, and polar moment of inertia) are shown in Table 2. No differences were detected in the endpoints measured. The effects of ethanol on bone mineralization (mineral/matrix, crystallinity/maturity, and CO32− v1/ PO43− v1) and mechanical properties (ultimate force, total work, ultimate stress, modulus, and toughness) are also shown in Table 2. No differences were detected in any of the endpoints measured. Additional bone mechanical property results, all of which showed no significant effects of ethanol, are presented in Supplemental Table 1.

Table 2.

Effects of 6 months of voluntary ethanol consumption on tibia length, tibial bone mineral content (BMC) and areal bone mineral density (BMD), distal tibia cortical bone architecture, tibia mineralization, and tibia mechanical properties in male cynomolgus macaques.
Control Ethanol P value Test
 Tibia Length (mm) 150 ± 2 149 ± 1 0.990 Two-sample T-test
Densitometry
 Bone Area (cm2) 20.9 ± 0.6 22.0 ± 0.9 0.777 Welch’s Two-sample T-test
 BMC (g) 7.0 ± 0.2 7.8 ± 0.5 0.642 Welch’s Two-sample T-test
 BMD (g/cm2) 0.34 ± 0.00 0.35 ± 0.01 0.777 Welch’s Two-sample T-test
microComputed Tomography
 Total volume (mm3) 58.7 ± 2.8 58.7 ± 1.7 0.998 Two-sample T-test
 Cortical volume (mm3) 40.5 ± 1.4 40.0 ± 1.0 0.990 Two-sample T-test
 Marrow volume (mm3) 18.3 ± 1.6 18.8 ± 1.3 0.990 Two-sample T-test
 Cortical thickness (μm) 1885 ± 35 1868 ± 48 0.990 Two-sample T-test
 Ipolar (mm4) 506 ± 43 499 ± 27 0.990 Two-sample T-test
Raman Spectroscopy
 Mineral/Matrix (PO43− v1 / Amide I) 3.77 ± 0.13 3.73 ± 0.05 0.990 Two-sample T-test
 Crystallinity/Maturity 0.051 ± 0.001 0.051 ± 0.0001 0.990 Welch’s Two-sample T-test
 CO32− v1 / PO43− v1 0.289 ± 0.005 0.284 ± 0.002 0.777 Welch’s Two-sample T-test
Mechanical Properties
 Ultimate Force (N) 865 ± 44 881 ± 37 0.990 Two-sample T-test
 Total Work (mJ) 3772 ± 405 3985 ± 230 0.990 Two-sample T-test
 Ultimate Stress (MPa) 391 ± 23 408 ± 16 0.990 Two-sample T-test
 Modulus (GPa) 83 ± 4 83 ± 4 0.998 Two-sample T-test
 Toughness (MPa) 4.3 ± 0.4 4.7 ± 0.2 0.779 Two-sample T-test
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Data are mean ± SE, n = 6 controls and 16–17 ethanol-consuming monkeys

4. Discussion

We investigated the effects of 6 months of daily voluntary ethanol consumption on bone density, cortical bone architecture, mechanics, mineralization, and intracortical bone porosity in tibia of young adult male cynomolgus macaques. An average daily ethanol intake of 2.3 ± 0.2 g/kg for 6 months, resulting in average daily BEC of 90 ± 12 mg/dl, led to a significant decrease in distal tibia intracortical bone porosity and a strong tendency for a decrease in plasma CTX. In contrast, ethanol consumption had no effect on tibial BMC or areal BMD, nor on cortical architecture, mineralization, or mechanical properties in the tibial diaphysis.

The present analysis complements and extends earlier studies investigating the effects of 12 and 20 months of voluntary ethanol consumption on tibia of young adult male rhesus macaques under the same housing conditions and alcohol protocol (Gaddini et al., 2015, Shin et al., 2024). In the rhesus studies, open-access drinking was associated with a reduction in intracortical bone porosity, which was attributed to near complete suppression of initiation of new secondary osteons (Gaddini et al., 2015). Osteons that were in the process of forming prior to introduction of alcohol continued to develop and were completed by the end of study. This likely occurred because the typical bone matrix formation and mineralization interval in the bone remodeling cycle of rhesus macaques, lasting ~2 months, was much shorter than the intervals of alcohol treatment. The present study refines the time course for the skeletal effects of alcohol on cortical bone by reducing open-access to alcohol to 6 months, a much shorter interval than previously examined (12 and 20 months).

Six months of open-access drinking in the cynomolgus macaques resulted in a reduction in intracortical porosity nearly identical to that observed following 12 months of open-access drinking in rhesus macaques. This finding suggests that the effects of alcohol on secondary osteons occurs quickly. Although we detected a dose-response effect of alcohol on cortical porosity in the current study, even the lowest levels of alcohol consumption by these monkeys appeared sufficient to reduce porosity.

Inhibition of initiation of new osteons is not unique to alcohol consumption. Pharmaceuticals such as bisphosphonates and estrogens slow bone loss associated with menopause-induced elevated bone turnover by this mechanism (Reid, 2008). A reduction in the rate of initiation of intracortical bone remodeling may be beneficial when bone turnover is excessively elevated (e.g., menopause). However, over suppression is likely to be detrimental. Because bone remodeling is essential to repair fatigue microdamage that accumulates during normal activity and reduce overall mean tissue age (Gabet and Bab, 2011, Burr et al., 1997), prolonged suppression could ultimately result in an increase in fracture risk (Seeman and Martin, 2019, Seeman, 2007, Ott et al., 2021).

The cortical bone turnover rate in the tibia of young adult male rhesus macaques is ~ 5 %/year (Gaddini et al., 2015). Thus, our finding that 6 months of open-access alcohol consumption did not result in changes in total bone mass and mechanical properties is not surprising. There was a tendency for a decrease (compared to baseline) in plasma CTX, a marker of bone resorption, in the alcohol-consuming monkeys. This finding concurs with a prior study performed in rhesus macaques (Benton et al., 2022) and is consistent with reduced bone turnover. The effects of alcohol on bone resorption in humans are less clear, with increases, no change and decreases reported (Chappard et al., 1991, Bikle et al., 1993, Diamond et al., 1989a, Diez et al., 1994, Pepersack et al., 1992, Nyquist et al., 1996). The explanation for the variability is not certain but may be due, at least in part, to comorbidities common to humans with alcohol use disorder (Castillo-Carniglia et al., 2019, Holst et al., 2017).

Chronic heavy alcohol consumption in humans is often associated with reduced biochemical markers of global bone formation (Nyquist et al., 1996, Rapuri et al., 2000, Diamond et al., 1989a, Diamond et al., 1989b). Analyses of human transiliac biopsies in chronic heavy alcohol consumers show reduced cancellous bone formation (Bikle et al., 1985, Bikle et al., 1993). In the present study, we did not see an effect of ethanol consumption on plasma osteocalcin levels. While we and others have consistently observed alcohol-induced decreases in osteocalcin gene expression and serum levels of osteocalcin in young rats where ethanol is a potent inhibitor of bone growth and accrual (Wagner et al., 2019, Turner et al., 2001a, Turner et al., 1998, Dyer et al., 1998, Diez et al., 1997, Peng et al., 1991), variable results have been reported in young adult monkeys (Benton et al., 2022).

Moderate alcohol consumption in humans is associated with higher DHEAS levels (Sacks et al., 2018), while cortisol appears to be low in chronic heavy alcohol consuming humans (King et al., 2011) and rhesus macaques (Jimenez and Grant, 2017). Abstinence from chronic heavy drinking results in an exaggerated cortisol response in humans (Sinha et al., 2011) and rhesus macaques (Allen et al., 2018). Both hormones are produced in adrenal glands and chronic elevation of cortisol is a well-established risk factor for reduced bone formation and bone loss (Suarez-Bregua et al., 2018). In contrast, DHEAS supplementation was shown to increase osteocalcin levels in humans (Papierska et al., 2012) and attenuate ovariectomy-induced bone loss in rats (Turner et al., 1990). Thus, it is plausible that changes in release of one or both of these hormones contributes to the skeletal response to alcohol. Whereas DHEAS levels at necropsy decreased compared to baseline in controls in the current study, this decline did not occur in monkeys that consumed alcohol.

Aging is the most important risk factor for low trauma fractures (Office of the Surgeon General (US), 2004). This is due, in part, to an age-related decrease in BMD and an increase in fall frequency (Bouxsein et al., 2019). However, there is also evidence that deterioration in bone quality may contribute to the age-associated decline in bone strength (Burr, 2019). Controlling for BMD and fall frequency, chronic alcohol abusers exhibit higher rates of fracture than the general population (Berg et al., 2008). This suggests that heavy alcohol consumption can reduce the structural integrity of the skeleton by a BMD-independent mechanism. Thus, chronic excessive alcohol consumption for long intervals may exaggerate bone loss associated with aging and other risk factors, including poor diet, physical inactivity, and chronic disease (Kelsey and Samelson, 2009).

Chronic alcohol abuse may influence vitamin D metabolism and consequently reduce intestinal calcium absorption which in turn could influence bone mineralization (Shankar et al., 2008, Turner et al., 1988). We did not measure vitamin D metabolites or calcium absorption. However, 12 months of open access to alcohol in young adult male rhesus macaques had no effect on plasma vitamin D levels (Gaddini et al., 2015). Additionally, in the present study we did not detect alcohol-induced changes in relative bone matrix mineralization by Raman spectroscopy, indicating that the hypothesized alcohol-induced decrease in bone quality is not likely due to a major change in mineralization. Taken together, these findings fail to support vitamin D as playing a causal role in a putative alcohol-induced defect in bone quality.

Limitations of this study include relatively small sample size, particularly for the controls, and a sole focus on tibia of young adult males. The small sample size limited the statistical power to detect differences in the bone architectural endpoints when controls are compared to monkeys having free access to alcohol. However, alcohol consumption was a continuum, ranging from 0 to over 3.5 g/kg/d, and a significant ethanol dose-dependent decrease in intracortical porosity was detected. We focused on young adult males in our model as chronic heavy alcohol consumption in humans is highest in young adult men (Substance Abuse and Mental Health Services Administration, 2014) and young adult male macaques (Helms et al., 2014). However, females and other age groups need to be evaluated for a more comprehensive understanding of the skeletal effects of alcohol across the lifespan. In addition, cortical bone sites in the axial skeleton should be evaluated in future studies.

In summary, the present evaluation performed in young adult male cynomolgus macaques demonstrates that 6 months of chronic heavy alcohol consumption resulted in a dose-response reduction in cortical bone porosity, likely due to suppressed intracortical bone remodeling, but did not alter other indices of bone quality. This finding demonstrates that even a relatively short (6 months) interval of heavy alcohol consumption may suppress the initiation of new osteons. Most low trauma fractures occur later in life. Our results suggest that by suppressing repair of fatigue microdamage to bone matrix or other outcomes linked to mean tissue age, decades of heavy alcohol consumption, common in humans, may lower bone quality. If our interpretation is correct, this mechanism explains, at least in part, why chronic alcohol abuse is associated with increased fracture risk, even when BMD is normal.

Supplementary Material

MMC1

Highlights.

  • Chronic heavy alcohol consumption is a risk factor for low trauma bone fractures.

  • We assessed effects of 6 mo of voluntary ethanol intake on cortical bone in monkeys.

  • Ethanol had no effect on tibia density, mechanical properties, or mineralization.

  • Ethanol resulted in a dose-dependent reduction in intracortical porosity.

  • Long-term suppressed intracortical remodeling may result in decreased bone quality.

Support:

This work was supported by grants from the National Institutes of Health (AA026289, AA013510, AA010760, AA019431) and the National Institute of Food and Agriculture – Agricultural Experimental Station Multi-state [W4002] and Oregon Agricultural Experiment Station [OR00735]. The Endocrine Technologies Core at the Oregon National Primate Research Center was supported by the National Institutes of Health (P51OD011092).

Footnotes

CRediT authorship contribution statement

Amida H. Kuah: Investigation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Lara H. Sattgast: Investigation, Writing – Review & Editing

Kathleen A. Grant: Conceptualization, Funding, Methodology, Project Administration, Resources, Supervision, Writing – Review & Editing

Steven W. Gonzales: Investigation, Writing – Review & Editing

Rupak Khadka: Investigation, Writing – Review & Editing

John G. Damrath: Investigation, Writing – Review & Editing

Matthew R. Allen: Resources, Writing – Review & Editing

David B. Burr: Resources, Writing – Review & Editing

Joseph M. Wallace: Resources, Writing – Review & Editing

Gianni F. Maddalozzo: Investigation, Writing – Review & Editing

Mary Lauren Benton: Data Curation, Writing – Review & Editing

Laura M. Beaver: Visualization, Data Curation, Writing – Review & Editing

Adam J. Branscum: Formal Analysis, Writing – Review & Editing

Russell T. Turner: Conceptualization, Investigation, Writing - Original Draft, Writing - Review & Editing

Urszula T. Iwaniec: Conceptualization, Funding, Project Administration, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing - Review & Editing

Declarations of interest: The authors have nothing to declare.

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