# Unveiling the hidden impact of long-term metal-rich volcanic pollution on male reproductive functions using isotope metallomics

Lucie Sauzéat, Mélanie Moreira, Hélène Holota, Claude Beaudoin, David H Volle

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## To cite this version:

Lucie Sauzéat, Mélanie Moreira, Hélène Holota, Claude Beaudoin, David H Volle. Unveiling the hidden impact of long-term metal-rich volcanic pollution on male reproductive functions using isotope metallomics. Environment International, 2025, 198, pp.109388. 10.1016/j.envint.2025.109388. hal-05263716

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# Unveiling the hidden impact of long-term metal-rich volcanic pollution on male reproductive functions using isotope metallomics

Lucie Sauzéat<sup>a,b,*</sup> <sup> ID </sup>, Mélanie Moreira<sup>a</sup>, Hélène Holota<sup>b</sup>, Claude Beaudoin<sup>b</sup>, David H. Volle<sup>b ID</sup>

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## ARTICLE INFO

Editor: Adrian Covaci

Keywords:

Metal-rich ash

Isotope metallomics

Oxidative stress

Male infertility

## ABSTRACT

Volcanic eruptions release particles in a range of sizes that can chronically affect the health of communities within tens of kilometers of the volcano. Many years after an eruption, resuspension of volcanic ash can exacerbate the health impact of primary eruptive events. So far, our global understanding of the health effects triggered by chronic exposure to volcanic particles at the whole-body scale is limited. Recently, it has been shown that mice chronically exposed to metal-rich volcanic ash deposits present metallome deregulations associated with pathophysiological changes. These deregulations preferentially impact the reproductive functions, questioning about the impact of ash on fertility. This work aims to further assess the mechanisms driving the ash-related fertility disorders and develop predictive biomarkers. For that, elemental concentrations and Cu-Zn-Fe isotope measurements coupled to metabolomic, proteomic and transcriptomic analyses were measured in blood, liver and two organs of the male reproductive system (testis, seminal vesicle). The samples were collected on wild-type and mice exposed over two months to volcanic ash. Mice exposed to ash are characterized by (i) significant metallomic deregulations, (ii) higher oxidative stress correlating with isotopic variations of redox-sensitive elements and (iii) testicular and hepatic alterations, marked by gains in organ mass, hepatic lipid accumulation and circulating bile acids overload, all of which might exacerbate testicular defects. Together, these results demonstrate that prolonged exposure to metal-rich ash is a threat for male reproduction and that investigating redox-sensitive isotopes might help identifying early signs of oxidative stress-related testicular injuries, with future implications for hepato-testicular disease prevention.

## 1. Introduction

During eruptive phases, volcanoes release hazardous contaminants including metal-rich gases (Ilyinskaya, 2021) and ash (Rodriguez-Espí-nosa, 2015), that can affect environmental (e.g., agricultural soils (Carrera-Beltrán, 2024), water (Stewart, 2020) and corals (Förster, 2024)) and human health (Baxter et al., 1983; Horwell and Baxter, 2006; Stewart, 2022; Camarinho et al., 2013). For example, people exposed to volcanogenic emissions, such as ash, are for example at higher risk of developing (cardio) respiratory injuries (Stewart, 2022) sustained by pathological lung injuries (e.g., decrease alveolar space, alteration of lung structural functionality, fibrosis) (Aguilera, 2018; Green, 1981). Volcanic ash is also found to provoke lung inflammatory reactions in animal models (Lee and Richards, 2004) and weak pro-inflammatory reactions in bronchial epithelial and alveolar cell culture models (Damby, 2018; Eychenne, 2022), evidencing the aggravating factor of

ash for acute respiratory diseases (Horwell and Baxter, 2006; Horwell, 2007; Horwell et al., 2007; Damby et al., 2016; Horwell et al., 2003). People living in volcanogenic environments are also most prone to birth outcomes and mental health sequelae (see Stewart, 2022 for review) and might also present higher incidence of gastrointestinal and thyroid cancers as are observed in Sicily (Pellegriti, 2009; Malandrino, 2020). Exposure to volcanogenic emissions is also associated with a higher risk of DNA damage in human buccal epithelial cells (Rodrigues et al., 2012) and worsening of mucosal inflammation in mice (Orsini Delgado, 2022). More recently, interdisciplinary studies performed on mice demonstrated that volcanically active environments, mimicked in the laboratory by chronic exposure to resuspended volcanic ash via multiple exposure pathways (oral ingestion and mouth/nose inhalation), favor the onset of testicular disorders that might, over time, contribute to the onset of hepato/testicular dysfunctions in rodent model (Ferreira et al., 2015; Sauzéat, 2022). Altogether, these results surfaced complex

<footnote><sup>a</sup> Université Clermont Auvergne, CNRS, IRD, OPGC, Laboratoire Magmas et Volcans, F-63000 Clermont-Ferrand, France</footnote>

<footnote><sup>b</sup> Université Clermont Auvergne, CNRS UMR 6293, Inserm U1103, Institut de Génétique, Reproduction et Développement, F-63000 Clermont-Ferrand, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: lucie.sauzeat@uca.fr (L. Sauzéat).</footnote>
adverse effects of volcanogenic environmental pollutants, and suggests that metal-rich pollution might alter male reproduction functions, possibly accounting for the ~20 % unexplained incidence of idiopathic infertile males reported in the worldwide male population (Agarwal, 2021). So far, neither the biological functions nor the mechanistic pathways impacted by chronic exposure to resuspended volcanic soil particles by multiple pathways at the whole-body scale, and more particularly on the male reproductive system have been elucidated, impacted our capacity to provide adequate care for exposed populations.

Another important question relies on the ash toxicity parameters. Volcanic ash are physical contaminants composed of crystalline and amorphous silicate components varying in chemical composition, morphology, mineralogy and surface reactivity. Ash can also contain variable inhalable, respirable (<100 µm and <10 µm) and oral (<250 µm) material (Horwell, 2007; Horwell, 2024). All these physicochemical properties, spatio-temporally variable from one volcanic setting to another and commonly quantified for health hazards assessment (Horwell, 2024), may account, either independently or in a synergistic effect, for the particles 'toxicity (Horwell and Baxter, 2006; Horwell et al., 2003; www.ivhnh.org). The presence of chemicals and pathogens like microorganisms on geogenic particles can also increase their bio-reactivity (Tomašek, 2025). Ash enriched in (pure) crystalline silica polymorph (Damby et al., 2016) or covered by reactive surface pollutants like anthropogenic (diesel exhaust) and organic (polycyclic aromatic hydrocarbons) pollutants (Tomašek, 2021; Tomašek, 2016; Tomašek, 2018) might for example be more toxic than pollutant-free or crystalline silica-poor ash although no consensus exists so far. Surface reactivity, driven by iron-induced hydroxyl radical (Horwell et al., 2007) might also contribute to ash toxicity. More recently, new health-metrics, such as the particles' oxidative potential (Daellenbach, 2020) partly attributable to the metal fraction of a particle (Vignal, 2017) has emerged while other parameters, like heavy metals, have been under scrutiny for several years. Though vital for the organism, metal (loid)s (e.g., Cu, Zn, Fe, Se) are highly harmful if transferred to and present in excess within the organism (Squitti et al., 2014). Due to its high metal (loid)s content (Rodriguez-Espinosa, 2015; Nanzyo et al., 2002) volcanic ash is a major source of metal contamination. For example, direct impacts are reported on soil's micronutrients and heavy-metal modulation content which is of importance to agriculture and the food chain (Carrera-Beltrán, 2024). Adverse side effects are also reported on living organisms, such as invertebrates (earthworms) (Amaral et al., 2006) and vertebrates (mice and humans) (Malandrino, 2020; Ferreira et al., 2015; Sauzéat, 2022; Amaral et al., 2007; Varrica et al., 2014) living in volcanically active environments, in which the accumulation of heavy metals in different tissues and organs has been demonstrated. In humans, this for example translates into a significant metal accumulation in urine (Malandrino, 2020) and hairs (Varrica et al., 2014) as reported in people living in the volcanically active province of Catania (eastern Sicily), with values two- or more times higher than in residents in a control group living in a non-volcanic area. Similar metalomic deregulations are observed in mice exposed over one month to resuspended volcanic ash deposits, as shown for example by heavy metal accumulation in testis (e.g., Fe, Zn, Cu) (Sauzéat, 2022). Note that these metalomic variations are often associated with concomitant health disorders like higher cancer incidence in humans (Malandrino, 2020) or early testicular defects in rodents (Ferreira et al., 2015; Sauzéat, 2022). This brings into question the possible carcinogenic effects and/or the role as infertility mediators of these potentially toxic elements (e.g., heavy metals) released by volcanic ash and absorbed by the organism. Beyond this dose effect commonly assessed by elemental concentrations, metal isotopic compositions have recently emerged as an important but still underexplored health metric. A pioneering study recently demonstrated that mice chronically exposed over one month to laboratory-crushed volcanic particles present significant Cu and Zn isotopic variations that may mirror hepato-testicular alterations (Sauzéat, 2022), but the timing and the factors governing the isotopic fractionation remain

unclear. Thus knowledge gaps about whether chronic exposure to metal-rich ash can trigger the development of severe diseases, especially testicular alterations, and regarding the growing but uncharted interest of state-of-the art isotopic measurements remain. This study was hence designed to (i) assess how chronic exposure to metal-rich volcanic ash contribute to testicular damages that may, over time, account for hypofertility and (ii) disentangle the ash-related isotopic changes to better investigate the utility of metal stable isotopes as future unconventional biomarkers in volcanogenic pollution context. We employ a transdisciplinary approach at the interface of volcanology, biology and isotope geochemistry, combining high precision chemical analyses (major and trace element concentrations), state-of-the art Cu, Zn and Fe isotope measurements in tandem with organ-specific biological parameters performed on isogenic mice (C57BL/6J) exposed to volcanic products from La Soufrière de Guadeloupe (Eastern Caribbean), identified as a potentially at-risk reactivating volcanic system (Moretti, 2020). Investigations were also carried out to better assess the inter-organ dialogue, especially between liver and testes. Liver diseases and male fertility disorders are linked, as evidenced in mice and humans (Mooradian et al., 1985), but the mechanisms involved in maintaining the hepato-testicular feedback loop has not been fully studied so far, and even less in volcanic context. A better understanding of the hepato-testicular interconnectedness is thus crucial to better address reproductive disorders preferentially affecting volcanic populations. Compared to our previous proof-of-concept study (Sauzéat, 2022), which was limited to one month's exposure, this study aims to get closer to chronic exposure conditions. For that, the time of exposure to volcanic ash was increased from one to two months. This study also goes one step further by investigating the mechanistic pathways likely accounting for the observed testicular alterations, including liver-testis dialogue. This study is also pioneering in the measurement of iron isotopic compositions in mice chronically exposed to volcanogenic pollutants. In comparison with the Cu and Zn isotope ratios and combined with biological data, this more complete and holistic approach bring meaningful information on the timing and factors accounting for the isotopic fractionations; an essential prerequisite for the development of redox-sensitive isotopes biomarkers under environmental volcanic pollution.

## 2. Materials and methods

### 2.1. Ethics statement

This study was conducted following current regulations and standards approved by the 'Institut National de la Santé et de la Recherche Médicale Animal Care Committee and by the animal care committee (CE2EA Auvergne; APAFIS#33604-2021102616039953 v2).

### 2.2. Animals & biological samples collection

In this study, we use the C57BL/6J mice as model species. For comparison with previous studies (Sauzéat, 2022), only males were considered. Mice were purchased from Charles River Laboratories (France). The protocols for volcanic ash exposure and mice sample collections are similar to the one previously published by Sauzéat (2022) except the time of exposure is doubled. Briefly, mice were housed in temperature, pressure and humidity-controlled rooms (22 °C, humidity <50 %), with 12-hour light/dark cycles. Mice had ad libitum access to food and water. Five-month-old mice (VAF/PLUS (SPF) mice, free of rodent pathogens) were exposed to volcanic ash (exposed group, n = 6) from La Soufrière de Guadeloupe volcano over ten weeks compared to a control group (no ash, n = 6 per group). For that, all the cages (500 cm² EasyCage® – Allentown) were filled with 100 g of corn cob litter (NestPak®) and volcanic ash (200 g of litter for 100 g of ash) for the exposed group. The complete procedure requires a total of 600 g of ash, which might be a limiting factor when considering duplicate
experiments and/or limited access to volcanic ash but which can be
easily circumvented by adapting the ash/litter ratios. To assess temporal
variations and compare our data with literature data, we use the same
ash samples previously used and characterized by Sauzéat et al.,
(Sauzéat, 2022) i.e.; a lab-crushed dacitic bulk ash (<2 mm) with a
median grain size of ~100 µm. Note that the bulk (<2 mm) ash sample,
comprising both the sub-100 µm inhalable and the sub-250 µm oral
particles has been used instead of isolated oral (<250 µm) or inhalable
(<100 µm) size fractions to avoid forcing an absorption pathway,
consider the synergistic and simultaneous inhalation/oral exposure
pathway and to take into account the particles interaction affecting
resuspension processes and thus controlling mammals' exposure con-
ditions, with coarser particles (>100 µm) playing a significant role in
fine particles production and resuspension processes (Fries, 2023; Gil-
llette et al., 1974). Body weight as well as food and water consumption
were then assessed over time exposure (Supp. Table S1). After ten weeks
of exposure, blood, liver, testis and seminal vesicle were collected,
weighed (Supp. Table S2) and separated into two or three symmetrically
identical or biologically-relevant sections for histological (only liver),
biological and isotope metalomic measurements. For histology, organs
were first paraformaldehyde (PFA)-fixed and then embedded in paraffin.
For biology, organs and fluids were directly stored at -80 °C until
further biological processing while, for metalomic measurements, or-
gans were first freeze-dried for 48 h (Christ Alpha™ Freeze dryer 1-4
LD+). As metalomic measurements are highly prone to metal exoge-
nous contamination bias, careful precautions were taken during sample
collections. These included wearing appropriate vinyl gloves (Garçon,
2017), using ceramic-coated-stainless steel surgical scissors (Ceram-
aCut® scissors, Fine Surgical Instruments for research™) and collecting
samples on acid-clean Eppendorf tubes®.

2.3. Histology

Histological measurements were only performed on liver. This multi-
lobed organ, it is the only one large enough to perform simultaneously
histological, biological and chemical analyses. Once collected, livers
were paraformaldehyde (PFA)-fixed (4 %) for 24 h and embedded in
paraffin. Five µm thick sections were then prepared and stained with
hematoxylin and eosin (H&E) and Masson's Trichrome (TM). Histo-
logical analyses were performed using a Zeiss Axioscan Microscope Slide
Scanner.

2.4. Sperm count

As a proxy for semen analysis, epididymal sperm counts were
assessed in both, the tail and the head of the epididymis. Epididymes
were disrupted and placed in phosphate-buffered saline (PBS) solution
to ensure spermatozoa remobilization. Samples were then diluted in PBS
(50-fold and 100-fold dilution) and sperm counts were determined using
a Malassez slide by counting the number of spermatozoa present in the
grids. Two independent determinations (N = 2) were performed and
were considered satisfactory when the difference between counts was
<20 %. Otherwise, counting was repeated. Final concentrations were
obtained by multiplying the results by the dilution factor. Data are re-
ported in Supplementary Table S2.

2.5. Fertility test

Ten days before euthanasia, five males per group (ash and control
groups) were mated at night with C57BL/6J female (Charles River) for
10 days. These reproduction tests were performed in cages without
volcanic ash. Mating was monitored daily for the presence of vaginal
plugs. After 20 days of gestation, the mating efficiency was visually
inspected and the number of pups per litter was counted (Supp.
Table S2).

2.6. Endocrine investigations & metabolomics measurements

Steroids were extracted from frozen testes with 10 volumes of hex-
ane/diethylether/acetic acid (80:20:2, v/v) as previously described
(Volle, 2007). An average of 16 mg (dry weight) is used. Intratesticular
testosterone levels were then measured using a commercial ELISA kit
(DetectX®, Testosterone Enzyme Immunoassay Kit, K032-H, Arbor
Assays™) according to the manufacturer's recommendations. Metab-
olomics measurements include blood plasma and liver bile acids and
lipids (triglycerides and total cholesterol) as well as blood plasma
circulating glucose level. For liver analyses, metabolites (bile acids and
lipids) were first extracted from frozen samples (~55 mg) with a mixture
(1:5) of methanol (MeOH) and acetonitrile (ACN), identified as the most
suitable extraction solvents for bile acids in organs and as previously
described (Vega, 2015), while lipids were extracted using the Folch's
extraction procedure (2:1 chloroform-methanol v/v) (Volle, 2007). Bile
acids and lipids, including total cholesterol and triglycerides, as well as
glucose measurements were then conducted in extracting solution or
directly on blood plasma using enzyme-linked immunosorbent and
colorimetric assay kits, following the manufacturer's recommendations
(bile acids: catalog no.: 80470; CrystalChem, Inc., Downers Grove, IL;
glucose: Glucose RTU, 61269, Biomerieux SA, France and triglycerides:
Triglycerides FS*, DiaSys Diagnostic Systems GmbH, Holzheim, Ger-
many; cholesterol: Calbiochem®, Merck Millipore, catalog no.: 428901).
All data are reported in Supp. Table S2.

2.7. Oxidative stress proteomic markers

Oxidative stress (OS) markers including antioxidant enzymes activ-
ities (i.e., total superoxide dismutase (SOD), cytosolic SOD1, mito-
chondrial SOD2, extracellular/plasmatic SOD3, total glutathione
peroxidase (GPX) and catalase (CAT)), and lipid peroxidation products
(malondialdehyde (MDA)) were measured in serum, liver and testis
mouse tissue lysates using colorimetric activity kits and following the
manufacturer's recommendations (CAT: catalog no.: Eiacatc, Invi-
trogen, ThermoFisher Scientific; GPX: catalog no.: MAK437, Sigma-
Aldrich, Merck; SOD: catalog no.: EIASODC, Invitrogen, ThermoFisher
Scientific; MDA: catalog no.: MAK085, Sigma-Aldrich, Merck). All data
are reported in Supp. Table S3. Briefly, for organ analyses, antioxidant
enzymes were first extracted from frozen samples (~40 mg for liver and
~20 mg for testis) either with ice-cold phosphate buffer solution
(PBS1X) or specific assay buffers. Extracting solutions were then
centrifuge (from 1,500 to 14,000 g) to pellet any debris for 10 to 15 min
either at room temperature or at 4 °C depending on the specificity of the
reagents. Only the clear supernatant was used for the assays. For plasma
samples, no preliminary extraction steps are required. Assays are per-
formed directly on the concentrated or diluted samples according to the
manufacturer's recommendations. Unused residual supernatants were
immediately frozen at -80 °C for further analyses. However, because
antioxidants are very sensitive to their environmental conditions (tem-
perature, light, oxygen, and pH) several precautions were taken. This
includes freezing supernatant up to a maximum of one month (see
manufacturer's recommendations) and avoiding multiple freeze-thaw
cycles of frozen samples. As illustrated on Supp. Fig. S1, repeated
measurements of cytosolic SOD1 on the same liver extracting solution at
one-month intervals (i.e., successive freeze-thaw cycles), can signifi-
cantly bias the measurement of SOD1 activity by more than a two-fold
increase.

2.8. *Quantitative reverse transcription polymerase chain reaction (RT-qPCR)*

RNA from mouse liver and testis samples were isolated using the NucleoSpin® RNA protocol (Macherey-Nagel, 740955, Hoerdt, France, ref: 740955.250). cDNA was synthesized from total RNA with the MMLV and random hexamer primers (Promega, Charbonnières-les-Bains,
France). The real-time PCR measurement of individual cDNA was performed using SYBR green dye (Master mix Plus for SYBR Assay, Takara Bio) to measure duplex DNA formation with the Mastercycler® Eppendorf-Realplex system. For each experiment, standard curves were generated with cDNA from liver and testis samples collected on control or mice exposed to volcanic ash. The results were analyzed using the ΔΔct method and data are reported in Supp. Table S4.

## 2.9. Metallomics measurements

Metallomics is a rapidly evolving field of bio-metal research that refers to the entirety of measurements aimed at quantifying metal- and metalloid species present in a biological system and defined as to their identity and/or quantity (Mahan et al., 2020). This includes major and trace element concentrations as well as isotopic compositions of biologically relevant metals such as copper (Cu), zinc (Zn) and iron (Fe) recently identified as the emerging field of isotope metallomics (Albarede, 2016).

### 2.9.1. Major and trace elements

All chemical analyses were carried out in clean laminar flow hoods using double-distilled acids as previously described. Freeze-dried liver, testis and seminal vesicle as well as blood were dissolved in a concentrated HNO₃-H₂O₂ mixture in Savillex beakers, first at room temperature for 24 h to avoid overpressure and loss by evaporation or leakage, and then at 100 °C for at least 72 h. After complete digestion, samples were first evaporated to dryness at 80 °C to avoid loss by volatilization of highly volatile elements like selenium (Se) (Vlastelic and Piro, 2022) and then, the residue was diluted in 10 mL HNO₃ 0.5 N. Weighed aliquots of this ‘mother’ solution were diluted with 2 % v/v HNO₃. Major and trace element concentrations were measured on ICP-AES (Agilent 5800) and QQQ-ICP-MS (Agilent 8900), respectively, at the Laboratoire Magmas et Volcans (LMV). The concentrations were calculated using calibrations curves performed with blank and different dilutions of multi-element standard solutions. For trace elements, indium (In, 2 ppb) was used as internal standard to assess instrumental drift. The validity and reproducibility of major and trace element concentrations is around 5 % (2sd) based on re-run analysis (bis) and complete duplicates of biological reference material (i.e., bovine liver, 1577c), except for some elements presenting analytical challenges (e.g., low elemental content close to the limit of detection, argon dimer interferences or low ionization efficiency) like arsenic (As), selenium (Se) and chromium (Cr) (Supp. Table S5). Major and trace element concentrations are all reported in µg/g (ppm) and ng/g (ppb) dry weight respectively in Supp. Table S5.

### 2.9.2. Cu, Zn and Fe isotopic compositions

Iron (Fe), zinc (Zn) and copper (Cu) isotopic compositions were carried out at the facilities of the Laboratoire Magmas et Volcans (LMV, France). Fully details of the analytical protocols adapted for biological samples can be found in Sauzéat (2022, 2021). Briefly, after digestion, the samples were evaporated to dryness and the residue was redissolved in a mixture of HCl (8 M) + H₂O₂ (0.001 %). Cu, Fe and Zn were then isolated by ion-exchange chromatography using 1 mL of AG-MP1 anion-exchange resin. A second chromatographic separation was applied to the Cu fraction following the same protocol to ensure the complete removal of sodium. Iron, copper and zinc isotopic compositions were then measured on a Thermo Scientific Neptune Plus™ MC-ICP-MS instrument (LMV, France) either in medium mass resolution for Cu and Zn isotope ratios or high mass resolution for Fe. The Cu, Zn and Fe isotope ratios are expressed in delta notation (δ⁶⁵Cu, δ⁶⁶Zn, δ⁶⁶Fe, per mil, ‰) relative to reference standards NIST SRM 976 for Cu, JMC 3-0749Lyon for Zn and IRMM14 as follows:

$$ \delta^{\text{xx}}A_{\text{sample}} = \left[ \frac{\left( \frac{^{\text{xx}}A}{^{\text{xy}}A} \right)_{\text{sample}}}{\left( \frac{^{\text{xx}}A}{^{\text{xy}}A} \right)_{\text{std}}} - 1 \right] * 10^3 $$

where $^{xx}$A and $^{xy}$A refer to $^{65}$Cu, $^{66}$Zn or $^{56}$Fe and $^{63}$Cu, $^{64}$Zn or $^{54}$Fe respectively.

Mass bias correction relied on a combination of external correction in a sample-standard bracketing approach (SSB) and internal mass fractionation (exponential law) using Zn, Cu and Ni elemental doping. The overall procedure led to total procedural blanks (n = 4) of 1.8 ng for Cu, 4 ng for Zn and 11 ng for Fe, which represents less than 1 % of the amount of each element present in the measurement solutions. The long-term precision of the results was assessed by sample re-run analysis and repeated measurements of the bracketing standards run every two samples. Sample accuracy was assessed by the measurement of certified biological reference material (bovine liver 1577c). The long-term external standard reproducibility (2sd) was better than 0.08 ‰ (n = 40) and our results of the certified reference 1577c material are in good agreement (average 1577c values (n > 14) < 0.03 ‰) difference with certified values for all isotope ratio measurements; see Supp. Table S5). Given our long-term precision and the accuracy obtained on reference material measurements, the two-standard deviation (2sd) analytical uncertainty adopted in this study for the Cu, Zn and Fe isotopic compositions is on average of ± 0.05 ‰. All data are reported in Supp. Table S5.

Note that the measurement of Cu, Zn and Fe isotopic compositions in small and low concentrated biological samples, like mice testis, may represent an analytical challenge. For testis, with an average Cu concentration of 6 ppm and an average mass of 18 mg (dry weight), one entire testis is needed to obtain the minimum amount of Cu required for precise isotopic measurements. Given that one testis is also required for biological analyses, each isotopic data point thus represents a distinct mice exposure assay. With the European animal welfare legislation that sets out legal requirements to implement the 3Rs principles of replacement, reduction and refinement, obtain many duplicates can thus be more complicated as usually carried out in biological studies, but in that case, data reliability is assessed by measuring replicates (bis) and certified standards as detailed above.

## 3. Results

### 3.1. Metallomic profiling and Cu, Zn and Fe isotopic footprint

Major and trace element concentrations, and Cu, Zn and Fe isotopic compositions (δ⁶⁵Cu, δ⁶⁶Zn, δ⁶⁶Fe respectively) were measured in the control and mice exposed to volcanic ash over two months. These results are reported in Supplementary Table S5 and those for the testes are shown in Fig. 1. To first explore the multivariate dataset and assess the global metallomic footprint, we performed a principal component analysis (PCA) that was then refined with biplot diagrams. As illustrated on Fig. 1A and as previously demonstrated in mice after one month of exposure (Sauzéat, 2022), mice daily exposed over two months to metal-rich volcanic ash showed altered metallome preferentially affecting the male reproductive system (testis). In the testes (Fig. 1A), this translates into higher concentrations of metals like Cu (from 6.3 ± 0.5 to 7.4 ± 0.4 ppm), Zn (from 104.9 ± 3.0 to 138.4 ± 3.7 ppm), Fe (from 178.8 ± 13.7 to 210.1 ± 6.1 ppm), Co (from 101.5 ± 7.9 to 126.5 ± 5.0 ppb), V (from 22.0 ± 3.0 to 31.4 ± 1.1 ppb) and Mn (from 1.57 ± 0.08 to 1.85 ± 0.10 ppm) as well as other non-metal elements such as selenium (Se) (from 3.05 ± 0.10 to 4.02 ± 0.16 ppm) which are potentially toxic if an excess is present within the organism (Fig. 1 and Supp. Table S5). The distribution and accumulation of metals, as demonstrated for Cu and Fe, are conservative patterns within the testes as illustrated by the similar trend and accumulation rate after one and two months of exposure (Fig. 1B). As shown in Fig. 1A, ash is the predominant factor in testicular chemical variations accounting for almost half of the observed variations (40 %),
<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Ash-related testicular metalomic deregulations. (A) Principal Component Analysis (PCA), (B) and (C) ash-related testicular heavy metal accumulation and redox-sensitive metal isotopic variations. In the PCA, the variables include the chemical concentrations of nineteen major and trace elements, the mass of the organ normalized to body weight as well as δ65Cu and δ66Zn values. All data were normalized, grey arrows are graphic representations of loading factors in the PC1 vs.PC2 space and the coordinates of each sample in the PCA space (i.e. sample scores) are shown by circle dots. Light and dark color points stand for control and exposed subjects, respectively. Data obtained on mice exposed over one month are from Sauzéat (2022). PCA was implemented in MATLAB™. In Fig. 1B, error bars represent two standard deviations (2sd) based on complete duplicate analyses and/or deviation from certified reference material (1577c, bovine liver).</figcaption></figure>

with age-related factors accounting for variations in the secondary component (17%). The testes have been suggested to be an organ highly prone to this volcanogenic pollution (Sauzéat, 2022). However, less pronounced metallome deregulations also extend to the rest of the organism. These include androgen-dependent organs like the seminal vesicle (cf Supp. Table S5), blood and liver, which is the most central organ in regulating systemic metal homeostasis (Supp. Fig. S2). However, in contrast to the testes for which significant ash-related variations are observed as early as after one month's exposure, the liver first maintains a relatively stable or even slightly *hypo-metallomic* internal state. This is illustrated by stable or slightly lower major and trace elemental contents after one month ash exposure, which is likely to be a consequence of its detoxification function. However, when the external forcing (i.e., ash exposure) lasts longer than 2 months, hepatic metal-lome alterations start to appear similar to those observed in the testes (Supp. Fig. S2). For example, copper levels rise from 15.8 ± 1.5 ppm to

18.0 ± 1.2 ppm (n = 6, SEM) after two months of exposure while it was first decreasing from 17.6 ± 0.9 ppm to 15.0 ± 0.4 ppm (n = 6, SEM) after a month. In addition to elemental concentrations, mice organs are also marked by significant metal stable isotopic changes. In the testes, this translates into isotopic variations of redox-sensitive elements including Cu and Fe (δ⁶⁵Cu rises from -0.76 ± 0.09 ‰ to -0.38 ± 0.07 ‰; p = 0.002 and δ⁵⁶Fe decreases from -0.35 ± 0.05 ‰ to -0.73 ± 0.07 ‰; p = 0.004) while the non-redox Zn isotopic system remains stable (δ⁶⁶Zn<sub>ctrl</sub> = +0.35 ± 0.02 ‰ vs δ⁶⁶Zn<sub>ash</sub> = +0.33 ± 0.04 ‰) (Fig. 1C). Similar variations are observed in the blood, liver and seminal vesicle i.e., stable δ⁶⁶Zn coupled with a δ⁶⁵Cu increase between +0.3 and +0.2 ‰ (see Supp. Table S5).

## 3.2. Testicular and hepatic alterations

Altered dietary habits, marked by lower but insignificant food and

<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Ash-related male hypofertility and testicular deregulations. (A) Numbers of pups per female mated with control or male exposed over 2 months to volcanic ash (n = 5 per group), (B) testicular testosterone level (pg.g⁻¹ wet mass) (n = 6 per group), (C) relative spermatozoa count in the epididymis head and tail in control and volcanic ash exposed mice. For sperm count, data include those of Sauzéat et al. (2022) (n = 12 per group), (D) testis weights (normalized to body weight). For all diagrams, error bars indicate SEM (i.e., standard error of the mean). Statistical p-value analyses are calculated as two-sided non-parametric Mann-Whitney *U* test implemented in MATLAB™.</figcaption></figure>
water consumption (mostly present in the first weeks of exposure), also emerged in mice exposed to volcanic ash but without noticeable impact on the body weight (Supp. Fig. S3). This pattern is similar to that observed in mice exposed over one month (Sauzéat, 2022). Mice exposed to volcanogenic pollutants also present increased signs of altered testicular and hepatic functions. This was first expressed by an increase in the mass of the liver and testis by 23 % and 15 % respectively, an increase that also extended to the seminal vesicle (+33 % increase) (Fig. 2D, Fig. 3A and Supp. Table S2). As shown in Fig. 2A, two month ash exposure also led to altered fertility, with a 20 % decrease in the number of pups per liter. This decrease is however not associated with a lower production of spermatozoa by the testis. As illustrated in Fig. 2C, testes are marked by a stable average number of spermatozoa in both the tail and the head of the epididymis after both one and two months of exposure, suggesting that ash exposure does not alter germ cell survival. No modification of intra-testicular testosterone was observed (Fig. 2B). In testes, gene expression levels of several androgen-dependent genes (*Pem*, *Cnx43* and *Osp1*) as well as genes involved in oxidative stress defense, cellular metal homeostasis, inflammation and bile acids synthesis were not significantly changed after two months of ash exposure. Only Gpx3 and Ccs genes; encoding for glutathione peroxidase (GPX) which is an antioxidant protein fighting against oxidative stress and a copper chaperone for SOD respectively; were slightly downregulated (Supp. Table S4) but not in a statistically significant way. Although not significant, the testicular gpx3 downregulation correlate with intra-

testicular metal overload (Supp. Fig. S4) likely attesting of metal toxicity. These results suggest that androgen-independent mechanisms likely underlie the observed altered fertility in mice chronically exposed to volcanic ash.

Liver histological analyses revealed the presence of lymphocyte infiltration (Fig. 3D) and hepatocellular vacuolization that might be the signs of fat deposits (Fig. 3E). These results are in line with the 66 % increase (p = 0.04) of liver triglycerides content and the 23 % mass increase (p = 0.03; Fig. 3A and C). Triglyceride levels remain however stable in blood plasma, like blood glucose, liver total cholesterol and bile acids (Supp. Table S2). Inversely, blood plasma is marked by significant increase of circulating bile acids (BAs) (p = 0.01; Fig. 3B), negatively correlated with the reduced fertility (Supp. Fig. S5). In the liver, gene expression levels of several genes encoding for antioxidant enzymes (Sod1-3, Gpx1-4; Cat) tend to be more or less downregulated (Supp. Table S4 and Fig. 3F). Altogether, these results show that the liver is marked by altered oxidative stress status and metabolic upregulation preferentially impacting lipid and bile acid metabolism, while carbohydrate metabolism seems to be preserved.

### 3.3. Oxidative stress

Several reports have revealed that exposure of organisms to environmental pollutants contributes to oxidative stress, a component of many diseases (Forman and Zhang, 2021) including testicular injuries

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Hepatic alterations. (A) liver mass normalized to body weight (wet weight), (B) circulating bile acids (µmol/L), (C) liver triglycerides (µg/g, wet weight), (D) & (E) liver histology (hematoxylin/Eosin staining) showing lymphocyte infiltration (C) and hepatocellular vacuolization (fat deposit) (D), (F) liver gene expression assessed by real-time RT-qPCR on multiple independent experiments ($1 \le N \le 3$; $n = 6$ per group for both control and ash exposed mice). Gene expression is normalized to the geometric mean of the expression of two housekeeping genes (β-actin and cyclophilin). Boxplot diagrams and statistical p-value (two-sided non-parametric Mann-Whitney U test) analyses were implemented in MATLAB™. * is for significant p-value $\le 0.05$.</figcaption></figure>
(Hug, 2023). By definition, oxidative stress is an imbalance between production of harmful oxidants (i.e., reactive oxygen species, ROS) and protective antioxidants. To determine whether volcanogenic ash contamination altered oxidative stress status, the gene expression and protein activity of the three main antioxidants enzymes (superoxide dismutase SOD, glutathione peroxidase GPX and catalase CAT) were measured in blood plasma, liver and testes to assess systemic and organ-specific oxidative stress. The MDA content of liver and testis tissues extract was also measured to assess lipid peroxidation; a secondary and indirect consequence of oxidative stress. Our results demonstrate that long-term volcanic ash exposure is able to alter the antioxidant response as first evidenced by the downregulation of several antioxidant genes in testes (Gpx3) and liver (Sod2, Gpx1 and Gpx4) (Supp. Table S4 and Fig. 3). These transcriptomic alterations superimpose to the slight but not significant protein activity decrease of mitochondrial SOD2, total GPX and CAT in liver, as well as cytosolic SOD1 and to a lesser extent CAT in testis, but far less pronounced than in liver (Supp. Table S3). Altogether, these proteomic and transcriptomic data point out an altered antioxidant response emplaced by the organism in favor of an increased oxidative stress at the whole-body scale likely more pronounced into the liver. Mice exposed to volcanic ash are also surprisingly marked by lower levels of triglyceride-normalized MDA level in liver, although this reduction is not visible in the testis tissue extracts (Supp. Table S3). MDA data should nevertheless be treated with caution. MDA lipid peroxidation markers positively covary with the content of triglycerides (data not shown) identified as major confounding factors. Higher level of lipids implies a greater amount of oxidizable substrates and a favorable scenario for the amplification of the chain reaction of lipid peroxidation. Triglyceride-normalized values should thus be considered to properly interpret the information provided by these markers (Pérez-Rodríguez, 2015). In addition, based on technical limitation and MDA assay kit non-specificity (Meagher and FitzGerald, 2000), the measurement of MDA as an index of lipid peroxidation has recently been highly questionable (Meagher and FitzGerald, 2000). Indeed, if the widely used “MDA test” utilizing thiobarbituric acid–reacting substance (TBARS) work reasonably well when applied to defined systems, such as liposomes and microsomes, its application to body fluids and tissue samples is more debated (Meagher and FitzGerald, 2000). The reliability of MDA as a representative oxidative stress marker is also questioned in this study by the lack of correlation with other robust oxidative stress markers such as SOD, CAT and GPX. As shown for example in the liver, while SOD, GPX and CAT protein activity are relatively well correlated ($r^2_{SOD2liv. vs CATliv} = 0.50; r^2_{SOD2liv. vs GPXliv} = 0.65; r^2_{MDAliv. vs GPXliv} = 0.52$), no correlation is observed with MDA level (e.g., $r^2_{MDAliv. vs GPXliv} = 0.14; r^2_{MDAliv. vs CATliv} = 0.09$) (data not shown) confirming that MDA are unspecific byproducts that are likely interfered by other compounds like sugars, amino acids, and bilirubin which are all, like MDA, also reactive toward TBARS (Meagher and FitzGerald, 2000).

# 4. Discussion

## 4.1. Metallome deregulation: From metal-rich environment to mice organs

The chronic health effects of long-duration eruptions have gone completely unmonitored and knowledge gaps remain about whether chronic exposure to metal-rich volcanic ash can trigger the development of global and severe disorders affecting functions beyond the respiratory system (Stewart, 2022), although few studies have suggested that this might be the case (Pellegriti, 2009; Malandrino, 2020; Sauzéat, 2022). As illustrated on Fig. 1A, mice exposed over two months (~five years on human scale) to metal-rich volcanic ash present significant metalomic deregulations preferentially affecting testes. This translates into both major and trace elements (Fig. 1B) as well as metal stable isotopic changes (Fig. 1C). For example, testes are marked by pronounced Cu and Fe accumulation from 6.3 ± 0.5 to 7.4 ± 0.4 ppm for Cu (x1.2) and from 178.8 ± 13.7 to 210.1 ± 6.1 ppm for Fe (x1.2) (Fig. 1B). These

variations are similar to the ones previously observed in mice exposed over one month, in which the average levels of Cu and Fe were similarly increased (on average from 6.4 to 7.6 ppm for Cu and from 159 to 208 ppm for Fe), with values about 1.2 times higher than in control mice (Sauzéat, 2022). Similar Fe increase (x1.2), although not significant, is also reported in worms captured at volcanogenic polluted sites compared to reference (no volcanic activity) sites (Amaral et al., 2006). After two months of exposure, mice are also marked by significant Pb (x4) and to a lesser extent Zn increase (x1.1 to x1.3) as shown in liver, testis and blood respectively. Like for Cu and Fe, these data are in line with data previously reported in mice and worms living in active volcanic area. For example, Zn increase of the same order of magnitude (x1.1 to x1.7) has been reported in the liver and tails of mice, as well as worms daily exposed to volcanogenic pollutants (Ferreira et al., 2015; Amaral et al., 2006; Amaral et al., 2007). Similarly, it has been found that the Pb concentration of liver collected on mice captured at volcanogenic polluted site is on average four times higher than mice from the area without volcanic activity, as observed in this study (Amaral et al., 2007). When comparing the data obtained on mice exposed over one versus two months to volcanic ash, beyond the concordant ash-related variations, a small offset is observed reflecting the age-dependent metallome composition (Fig. 1A). As previously evidence in humans (Jaouen, 2013), mice (Morel, 2022) and worms (Sauzéat et al., 2018), the age-dependent metallome fingerprint is a highly-conserved mechanism across different species. When comparing our results with recent mice and worms studies (Morel, 2022; Sauzéat et al., 2018), which measured a similar subset of the metallome in mouse organs and worms at different ages, age effects converged in all the studies despite very different timepoints, diets and species. For example, $\delta^{65}\text{Cu}$ of ~-0.3 ‰ to ~-0.6 ‰ has been observed during aging in liver mice (wild type) (Morel, 2022) and worms (C.elegans daf-2 mutants) (Sauzéat et al., 2018) respectively, in line with the ~-0.4 ‰ and ~-0.6 ‰ offset observed in this study between the liver and the blood of six (Sauzéat, 2022) and seven months-old mice (control mice; this study) respectively. Like blood and liver, testes are marked by similar age-related metallome variations as illustrated by the ~-0.6 ‰ drop between six and seven-month-old mice (this study). Taken together, these results confirm that age-related metallome changes is conservative over time, among several species, and at the scale of the whole body. Inversely, although the metallome of the liver is similarly marked by age-related variations, it is also characterized by more subtle and progressive ash-related temporal variations compared to the testes (Supp. Fig. S2A). After one month of exposure, the liver tends to maintain a relatively stable and/or hypo-regulate its metallome (Sauzéat, 2022), as shown for example by lower hepatic Cu content in mice exposed to volcanic ash (Supp. Fig. S2B), while metal (e.g., Cu) accumulation starts to appear after prolonged (two months) exposure (Supp. Fig. S2B). This progressive, inverse, although not significant, temporal metalomic transition might result from efficient metal detoxification (i.e., regulated metal homeostasis) to ongoing metal detoxification loss-of-function and liver metal dyshomeostasis, both signs of hepatic alterations (Costas-Rodríguez, 2015; Aramendía et al., 2013). More broadly, and in line with previous studies conducted on humans (Malandrino, 2020; Varrica et al., 2014), vertebrates (rodents) (Ferreira et al., 2015; Sauzéat, 2022; Amaral et al., 2007) and invertebrates (worms) (Amaral et al., 2006), these data confirm that long-term exposure to metal-rich volcanogenic pollutants contribute to significant organ-specific and isotopically-typified metallome deregulations at the entire body scale. Over time, these ash-related metallome deregulations might potentially account for diseases as suggested for cancers (Malandrino, 2020) and/or hepatosticular dysfunctions subsequently affecting male reproductive functions (Sauzéat, 2022). Note that not all volcanic systems have the same level of metal volcanogenic pollution with variability in the effects on the metallome and subsequent health effects. Hydrothermal fumarolic systems like La Soufrière de Guadeloupe have lower heavy metal emissions rates compared to magmatic emissions, but can still be
regarded as an important pollution source in the Lesser Antilles volcanic arc (Inostroza, 2023). In this study, the use of andesitic ash from La Soufrière de Guadeloupe might thus tend to under-evaluate the effects on the metallome deregulation.

## 4.2. Adverse effects of long-term metal-rich volcanic pollution on the male reproductive system: Is metal-induced oxidative stress a causal mechanism of ash-associated testicular disorders?

It is now well accepted that metal(loid)s (e.g., Cu, Zn, Fe, Se) are highly harmful if present in excess (i.e., above the recommended dietary allowance (RDA) with average RDA<sub>Cu</sub> ~1 mg, RDA<sub>Zn,Fe</sub> ~10 mg but variable as a function of the age, sexe, pregnancy and food practices) and subsequently accumulating within the organism (Squitti et al., 2014). Some typical, but non-exhaustive, pathologies marked by a disturbed metal homeostasis include for example neurological disorders; in which excess of metals like copper accelerate toxic protein aggregates (e.g., β-amyloid) and neurotoxicity (Gil-Bea et al., 2017; Barnham and Bush, 2014); cancer, with Cu excess stimulating proliferation of cancer cells and tumor growth (Ishida et al., 2013) and ageing, with excess micro-nutrients (Zn, Cu, Fe) altering age-related biological functions like the immune and the antioxidant performances (Mocchegiani, 2012). Heavy metal pollution has also been identified as an influential factor on fertility (López-Botella, 2021; Crocetto, 2023). In general, the toxicant's effects on male reproductive toxicity are complex, including oxidative stress, inflammation, apoptosis, and exocrine and endocrine disruption (López-Botella, 2021). Alteration of the spermatogenesis process, seminal quality and sperm motility as well as increased sperm DNA damages and a dysfunction in Sertoli cells (SCs) are, for example, several impacts of heavy metals on human male fertility (Chen, 2020). Heavy metals can also impair the hormonal regulation. In this latter case, heavy metals mimic hormonal functions being assimilated as endocrine-disrupting compounds (López-Botella, 2021).

In the present study, our data demonstrate that two months of exposure to metal-rich ash leads to altered testicular metallome composition (Fig. 1) with reduced male fertility as illustrated by fewer pups per litter in mice exposed to ash (~20 %) (Fig. 2A). This hypo-fertility developed along with higher testis weight (~15 %, Fig. 2D) but independently of testicular steroidogenesis repression and altered sperm count as showed by no modification of either intratesticular testosterone or of the number of spermatozoa in ash exposed mice (Fig. 2 B and C). Testis weight is a trait that has been associated with environmental contaminants-related reproductive efficiency across numerous species, attesting of pathophysiological intratesticular alterations (e.g., an increase in the number of germ cells, seminiferous tubule fluid content and tumor development) (Nava-Castro, 2019). Based on the literature data and in line with previous studies evidencing the adverse effects of volcanic pollutants on male reproduction, including spermatogenesis alterations (Sauzéat, 2022) concomitant of histopathological changes (e.g., disorganization of the germinal epithelium; reduction of spermatozoa in the lumen, enlargement of the interstitial spaces and decrease in the mean diameter of the seminiferous tubules), increased damage of seminiferous tubules and increased number of apoptotic spermatogenic cells (Ferreira et al., 2015; Sauzéat, 2022), the ash-associated testis weight increase might thus be an additional sign of testicular malfunction and confirmation of metal-rich ash exposure-mediated testicular toxicity. Overall, our results suggest that metals might act as modulators of the development of genital tract abnormalities preferentially impairing the exocrine spermatogenesis process likely in a sperm quality but not quantity-dependent manner. This echoes a recent study, that demonstrated that environmental-like pollutants impair male fertility due to germ cell dyshomeostasis and altered sperm quality (specifically histone retention) but not their quantity (Monrose, 2024).

The mechanism behind this metal-rich ash exposure-mediated testicular toxicity, might, in part, be attributed to oxidative stress which is a common precursor of several diseases (Forman and Zhang,

2021). Oxidative stress (OS) is defined as an imbalance between the production of reactive oxygen species (ROS) and antioxidant defences. Heavy metal ions can interact additively, synergistically or antagonistically by affecting each other's absorption, distribution and excretion (often referred to as a cocktail effect) and exert their toxicity by multiple mechanisms. Many metals, especially redox-active metals, are known to induce oxidative stress by (i) scavenging thiol groups, (ii) altering inflammatory response, (iii) impairing expression of oxidative stress-related genes or by (iv) directly enhancing reactive oxygen species (ROS) formation such as hydroxyl radical (HO'), superoxide radical (O₂⁻) or hydrogen peroxide (H₂O₂) via the Fenton-like reaction (Jomova and Valko, 2011). Metal-mediated formation of ROS can cause various modifications to DNA bases, proteins, membrane lipids and lipoproteins, identified as the main targets of ROS. Oxydative stress (OS) is a component of many diseases omnipresent in testicular injuries (Hug, 2023; López-Botella, 2021). As illustrated in Fig. 3F, ash-exposed animals display higher hepatic and, to a lesser extent, testicular dysregulated antioxidant systems, demonstrated by the general downward expression trend of genes coding for major antioxidants enzymes in liver (from most to least significant Gpx1, Sod2, Gpx4, Sod3, Gpx3, Cat) (Supp. Table S4) superimposed by slight antioxidant protein activity alteration (e.g., mitochondrial SOD2, CAT<sub>tot</sub>, GPX<sub>tot</sub> in liver) (Supp. Table S3). As antioxidant capacity is inversely proportional to oxidative stress and given the correlation observed between metal overload and gene expression, as observed in the tests (Supp. Fig. S4), these data indicate higher oxidative stress status in metal-rich ash-exposed mice. These data are in line with previous studies (Aguilera, 2018; Damby, 2018; Camarinho et al., 2021). Previous studies assessing the biological impact of volcanic ash on cell cultures demonstrated that volcanic ash exposure decrease the activity in superoxide dismutase by about 60 % (Aguilera, 2018) and involves the activation of mitochondrial stress pathways and of the inflammasome complex NLRP3 leading to the formation of reactive oxygen species (Aguilera, 2018; Damby, 2018), as illustrated in mice macrophage and fibroblast (McCoy) and human lung cancer (A549) cell lines. It was also found that mice chronically exposed to metal-rich hydrothermal volcanic emissions present increased levels of pulmonary oxidative stress (Camarinho et al., 2021). Altogether, these data confirm that the oxidative potential of metal-rich pollutants, including ash, is an environmental stressor contributing to adverse oxidative stress conditions. Compared to cell culture assays (Aguilera, 2018; Damby, 2018), by adopting a more complete in-vivo approach, this study goes one step further and highlights that ash-related OS likely operates over the entire body, with a more pronounced activity in the liver due to its major role in metal homeostasis and high metabolic rate that favor mitochondrial ROS formation.

In addition, the ash exposure-mediated testicular toxicity might also result from more complex and indirect organs crosstalk. Emerging pathogenic links have been highlighted between several organs such as evidenced within the (microbiota) gut-lung axis in the context of air pollution (Budden, 2017). Organ interconnectedness has also been observed for the liver-testis axis as illustrated by correlations between male fertility disorders and adverse conditions such as liver pathologies in humans and rodents (Mooradian et al., 1985; Durazzo, 2006). Among all factors that can be involved in the liver-testis dialog, bile acids (BAs) have been identified as potential mediators in the testicular physiopathologies (Baptissart, 2014; Baptissart, 2018; Sèdes, 2017). Bile acids have been demonstrated to alter male reproductive functions and decrease the number of pups per litter of a similar order of magnitude (20 % decrease) to what is observed in this study in the context of ash exposure (Fig. 2A) (Baptissart, 2014). Male infertility has been assigned to abnormalities of the germ cell lineage dependent on bile-acid-receptor signaling pathways (Baptissart, 2014; Baptissart, 2018). These effects of bile acids are mediated by specific receptor signaling pathways (Monrose, 2021) (e.g., Fxrα and Tgr5 receptors (Baptissart, 2018; Baptissart, 2016; Holota, 2020; Thirouard, 2022). Consistent with these reports, mice exposed to volcanic ash present significant hepatic
alterations as shown by an increased liver weight (Fig. 3A) implying
significant hepatic triglycerides accumulation (Fig. 3C) and moderate
histological alterations (e.g., hepatocellular vacuolization that might be
the sign of fat deposit, Fig. 3D & 3E), as well as higher levels of circu-
lating bile acids (Fig. 3B). These results, additionally supported by the
ash-related negative correlation observed between the number of pups
per litter with the increased plasma bile acid levels (Supp. Fig. S5),
demonstrate that, in addition to oxidative stress, hepatic alterations (e.
g., driven by bile acids overload) may also play a pathogenical role in
ash-related fertility disorders.

4.3. The future of isotope metallomics in diagnosing oxidative-stress related male hypofertility

In addition to altered elemental concentrations (cf part 4.1), long-
term volcanic ash exposure also impairs the testicular isotopic compo-
sitions of redox-sensitive metals i.e., Cu (δ⁶⁵Cu) and Fe (δ⁶⁶Fe).
Inversely, Zn isotopic compositions (δ⁶⁶Zn, a redox-inert element with
only one valence state), remains preserved over time (Fig. 1C). These
variations, at least for copper isotope ratios, extend to the rest of the
organism (i.e., liver, blood and seminal vesicle, Supp. Table S5). In
testes, this translates to a significant increase in δ⁶⁵Cu (about +0.4 ‰)
concomitant to a decrease δ⁶⁶Fe (about -0.4 ‰) (Fig. 1C) but stable
δ⁶⁶Zn. With the exception of δ⁶⁶Fe measurements, for which this study is
pioneering when considering volcanic health risk assessment, these
isotopic data are concordant with previously published data. For
example, after one month of exposure to volcanic ash, it was found that
the testicular zinc isotopic compositions were ranging between 0.35 ±
0.07 ‰ (1sd) in control mice versus 0.36 ± 0.05 ‰ (1sd) in exposed
mice, similarly to what observed in this study after two months of
exposure (i.e., δ⁶⁶Znctrl = 0.35 ± 0.04 ‰ (1sd) and δ⁶⁶Znash = 0.34 ±
0.10 ‰ (1sd)). Diet and/or exogeneous pollutants like ash are expected
to be the primary driver of the isotope compositions of the body (Tacail,
2023), although the biological processing of Cu, Zn and Fe is expected to
induce additional variabilities (Vanhaecke and Costas-Rodríguez,
2021). In this study, as mice experimental assays are performed under
controlled laboratory conditions, with identical diets between the con-
trol and exposed groups, diet cannot account for the observed Cu and Fe
isotopic changes. Similarly, if ash was the single, primary control on
isotopic variabilities, we would expect to observe Cu, Fe and Zn isotopic
variations with more pronounced changes for Cu and Zn due to their
relatively shorter turnover rates in the body compared to iron (Albarede,
2016). In contrast, mice exposed to volcanic ash present stable testicular
δ⁶⁶Zn (Fig. 1C and Supp. Table S5). The inverse temporal trend observed
for δ⁶⁵Cu, first decreasing after one month of exposure in testes, liver
and blood, and then increasing over the second month of exposure
(Supp. Fig. S6A), as well as the linear trend observed between δ⁶⁵Cu and
δ⁶⁶Fe, with the control group aligning on the same trend drawn by the
ash exposed group (Supp. Fig. S6B), also suggest that the Cu and Fe
isotopic signatures are not mainly in favor of an ash origin but rather
suggests an impaired metabolism of the redox-active bio-essential ele-
ments (i.e., Cu and Fe), expressed both under basal and more intensely
under pathophysiological conditions, likely by changing their redox
state. This hypothesis is consistent with stable δ⁶⁶Zn, as at the body
temperature like at Earth surface conditions, zinc does not undergo
changes in oxidation state (Moynier et al., 2017).

Stable isotopes are powerful tracers of physico-chemical processes, that first prove to be capable of unravelling Earths' history (Sauzéat et al., 2015). More recently, they have also proved to be potential predictors of metabolic health (Morel, 2022) and highly powerful for diseases' diagnosis and prognosis (Mahan et al., 2020; Albarede, 2016; Vanhaecke and Costas-Rodríguez, 2021; Sauzéat et al., 2018); although much remains to be learned on the timing and the exact mechanisms underpinning these isotopic shifts (Miaou and Tissot, 2023). In theory, mass-dependent stable isotope fractionation mainly depends on the bonding environment among isotopes and coexisting molecular species

which varies as a function of the bond strength. This is itself dependent
on the ionic charge (i.e., the oxidation state of the chemical elements)
and the coordination number of the ligand (Albarede, 2016) with bonds
involving high oxidation states (Fe³⁺, Cu²⁺) and sites with small coor-
dination numbers preferring heavy to light isotopes. As a result, proteins
with sulfur-rich cysteine ligands preferentially bound the light copper
isotope (⁶³Cu) while oxygen (glutamate, aspartate, lactate) and to a
lesser extent nitrogen (histidine) ligands favor the heavy isotope (⁶⁵Cu)
as in SOD (Albarede, 2016; Larner et al., 2019; Selden et al., 2024).
Alterations of the metal-binding environment, via for example the for-
mation of high-affinity metal-protein aggregates as suggested in
neurodegenerative disorders (Sauzéat et al., 2018; Moynier et al., 2017),
is thus one of the main biological processes accounting for metal stable
isotopic differences in biological systems. Additionally, inflammation
(Aranaz, 2022), selective transmembrane uptake (i.e., preferential up-
take of the light isotope) by specific metal transporter (e.g., CTR1 for Cu
modulated by Cu reduction) (Cadiou, 2017) and/or oxidative stress are
also likely to be important but still underexplored factors contributing to
redox-sensitive elements isotopic changes (Miaou and Tissot, 2023;
Balter, 2015; Bondanese, 2016; Telouk, 2022; Flórez et al., 2018; Ara-
naz, 2022). In this study, neither inflammatory processes nor impair-
ment of metal transporter uptake seems to be the main drivers of the Cu
and Fe isotopic changes observed in the testis, liver, blood and seminal
vesicle of mice chronically exposed to volcanic ash. As demonstrated by
transcriptomic measurements in the liver and testis, neither genes
encoding for metal transporters (e.g., Ctr1; Atp7a, Atp7b, Ccs) nor
inflammasomes (Casp1, Nlrp3) are significantly up or down-regulated
(Fig. 3F and Supp. Table S4). Conversely, and as discussed previously,
mice exposed to volcanic ash present an altered oxidative stress status,
with δ⁶⁵Cu and δ⁶⁶Fe significantly correlating with antioxidant markers
at both the transcriptomic and proteomic level (Fig. 4). As illustrated in
Fig. 4B and D, δ⁶⁵Cu increases and δ⁶⁶Fe decreases respectively with the
downregulation of antioxidant genes, as illustrated for Gpx3 in testis
(Fig. 4B) and mitochondrial Sod2 in liver (Fig. 4D). These Cu-Fe isotopic
correlations with oxidative stress markers are more pronounced in the
liver as supported by more significant Pearson correlation coefficients
and two-sided non-parametric Mann-Whitney *U* test p-values compared
to other body reservoirs (e.g., blood and testis) (Fig. 4A). In line with
previous studies (Balter, 2015; Bondanese, 2016; Telouk, 2022; Flórez
et al., 2018) which demonstrated that hypoxic conditions and/or in-
crease oxidative stress account for heavy copper enrichment in human
cell lines and liver tumor tissues, these results support an oxidative stress
(OS)-induced alteration of redox-active bio-essential elements (i.e., Cu
and Fe) bringing to light the potential of oxidative stress in isotope
fractionation. Measuring non-traditional redox-sensitive isotope proxies
might thus bring meaningful information in volcanic health hazard
assessment. Note that in testis, δ⁶⁵Cu also correlates with Cu:(Zn, Se)
ratios (r² = 0.5, Supp. Table S5) identified as oxidative stress
biochemical markers (Ozturk et al., 2013), additionally supporting a
causal link between redox-sensitive isotope fractionation and OS. The
mechanisms underpinning these OS-related isotopic shifts are not clear
yet but, based on literature data (Albarede, 2016; Selden et al., 2024);
this might include redox state changes and/or ROS-induced metal-
loproteins alterations. First demonstrated in geological samples, redox
transition (e.g., Cu(I) to Cu(II)), altered under oxidative conditions, in-
volves large isotopic fractionation (Moynier et al., 2017), with oxidized
cations being expected to favor heavy isotopes relative to their reduced
counterparts. Pro-oxidizing conditions favored under oxidative stress
can also alter the reversible oxido-reduction changes of Cu and Fe
regulating the catalytic cycle of antioxidant enzymes (e.g., reactions
catalyzed by SOD involve ping-pong conversion from Cu²⁺ to Cu⁺). In
turn, this might impact, in an isotopically-dependent manner, their
ability to correctly bind these specific molecules and subsequently their
antioxidant's efficiency. ROS, such as hydrogen peroxide (H₂O₂), are
also able to oxidize metal-ligand bonds like cysteine residues on target
metalloproteins (Lennicke and Cochemé, 2021). These oxidative
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** Oxidative-stress related redox-sensitive isotopic variations (A) Absolute person correlation coefficient between oxidative stress parameters and isotope metalomics data. Heatmap diagram and statistical p-value (two-sided non-parametric Mann-Whitney *U* test) analyses were implemented in MATLAB™. * is for significant p-value ≤ 0.05. (B) to (D) correlation between redox-sensitive isotope ratios and antioxidant gene expression (fold increase).</figcaption></figure>

modifications can similarly impair the biological activity of these metal-
binding proteins and, as metal-binding amino acid ligands differentially
fractionate metal-stable isotopes (Albarede, 2016; Selden et al., 2024),
this can subsequently contribute to significant metal-stable isotopic
variations. Among metalloproteins that might account for the concom-
itant altered δ⁶⁵Cu and δ⁵⁶Fe, metallothioneins (MTs), the superoxide
dismutase (SOD) (Larner et al., 2019; Selden et al., 2024) and/or the
ceruloplasmin (Cp), a major Cu-carrying and ferroxidase protein
essential for Fe metabolism enriched in ⁶³Cu (i.e., light δ⁶⁵Cu) (Tennant
et al., 2017) are potential candidates. But whatever the mechanisms, our
results evidenced that chronic ash exposure contributes to a pro-oxidant
environment translating into high oxidative stress levels in favor of
redox-sensitive metal isotope homeostasis alteration. Redox-sensitive
isotope proxies thus appear as promising OS biomarkers. Interestingly,
it has been suggested that the initial steps of the oxidation process might
first lead to an induction of antioxidant enzymes, whereas a decreased
antioxidant capacity is more likely the consequence of long-lasting
oxidative damage (Aranaz, 2022; Zanon-Moreno, 2008). Considering

that the antioxidant capacity is inversely proportional to oxidative
stress, and given that δ⁶⁵Cu increase and inverse δ⁵⁶Fe decrease mirror
high oxidative stress level (Fig. 4), the progressive establishment of
oxidative stress should theoretically first translate into initial δ⁶⁵Cu
decrease concomitant of δ⁵⁶Fe increase, and then evolve toward higher
δ⁶⁵Cu and lower δ⁵⁶Fe. Similar variations, at least for copper, are
observed in mice chronically exposed to volcanic ash with an isotopic
transition observed between one- and two-month exposure (Supp.
Fig. S6A). Under volcanogenic ash pollution, the initial drop in δ⁶⁵Cu
previously observed in liver mice after one month's exposure followed
by a rebound after two months of exposure might thus be interpreted as
the progressive and temporal onset of oxidative stress, with decreasing
δ⁶⁵Cu being the sign of the initial phase of oxidative stress while pro-
gressive δ⁶⁵Cu increase reflects long-lasting oxidative damages. Tem-
poral Cu and Fe isotopic changes are thus likely more relevant to
diagnose OS compared to punctual analyses that might be hampered
by individual variabilities. Applied to ash-related infertility disorders,
these emerging OS-related redox-sensitive isotopic biomarkers are
highly promising as male infertility remains poorly addressed. Routine and conventional diagnosis approaches (physical examination, serological test and semen analysis) are still insufficient (Agarwal, 2021; López-Botella, 2021). The development of novel and unconventional redox-sensitive stable isotope biomarkers may thus bring innovative and valuable information for human male fertility diagnosis in volcanic environments. Currently the measurement of testicular and/or hepatic δ⁶⁵Cu and δ⁵⁶Fe requires a biopsy, i.e., an invasive surgical intervention that may limit the scope of this marker. However, the significant correlations observed between δ⁶⁵Cu and δ⁵⁶Fe of organs (testis and liver) and blood (Fig. 4B) suggest that an assessment of the blood isotopic counterpart is likely to be an equally robust but far less invasive alternative.

## 5. Conclusions and perspectives

The chronic effects of volcanic pollutants, including volcanic ash, remain largely under-explored, which limits our understanding of the long-term volcanic ash-related risk at the organism scale. In this study, we demonstrate that after two months of exposure to metal-rich volcanic ash, mice exhibit significant metallome deregulations associated with pronounced hepatoo-testicular pathophysiological alterations. This mainly translates into higher oxidative stress, gains in organ mass, hepatic lipid accumulation and circulating bile acids overload, that all might account for fertility disorders over time. In volcanic environments, permanent low-level and multi-elemental volcanogenic pollution (cocktail effect) is thus of major concern that might contribute to chronic liver-testis axis impairment and subsequent male hypofertility. Based on unconventional yet state-of-the-art isotopic measurements, this study also demonstrates for the first time the emergence of redox-sensitive stable metal isotopes (δ⁶⁵Cu and δ⁵⁶Fe) as promising oxidative-stress (OS) biomarkers, with OS surfaced as a major factor involved in the ash exposure-mediated testicular toxicity. This offers a unique opportunity to probe oxidative stress-related infertility in volcanic environments. With the goal of biological and isotopic monitoring in humans, further validation is required on human subjects daily exposed to environmental volcanogenic pollutants. A better understanding of the mechanisms accounting for the oxidative stress-related isotopic changes as well as for the interconnectedness of the liver-testis organ system are required. This study also highlights how important coordinated and interdisciplinary efforts at the interface of volcanology, biology and isotope geochemistry are needed to assess and successfully prepare for managing health hazards associated with volcanic phenomena.

### CRediT authorship contribution statement

**Lucie Sauzéat:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Methodology, Investigation, Funding acquisition, Conceptualization. **Mélanie Moreira:** Formal analysis. **Hélène Holota:** Methodology, Formal analysis. **Claude Beaudoin:** Writing – review & editing, Funding acquisition, Conceptualization. **David H. Volle:** Writing – review & editing, Funding acquisition, Conceptualization.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Acknowledgements

We gratefully acknowledge Chantal Bosq, Abdel-Mouhcine Gannoun, Jean-Luc Piro and Claire Fonquernie of the Laboratoire Magmas and Volcans (UCA-LMV) for their assistance in the clean laboratory as well as during isotopic, trace and major element measurements

respectively. Thanks are also due to Sandrine Plantade and the entire research animal facility team for their help at the animal facility. We also gratefully thank the AniPath platform from the iGReD for histological analyses and David E. Jessop (UCA-OPGC-LMV, IPGP) for his invaluable help in sampling geological samples and helpful comments on the manuscript. This research was mainly financed by the French government IDEX-ISITE initiative 16-IDEX-0001 (CAP 20-25) of the Clermont Auvergne University (UCA) and was also supported by the French National Center for Scientific Research (CNRS), the French National Institute of Health and Medical Research (INSERM), the Plan Cancer-INSERM (C20010CS), the French League Against Cancer (R19037CC, Puy-de-Dôme committee) and the ANR (ANR-21-CE14-0083-02 & ANR-22-CE34-007-01). This is a Contribution N° 682 of the ClerVolc Program of Excellence of the International Research Center of Disaster Science and Sustainable Development.

## Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.org/10.1016/j.envint.2025.109388.

### Data availability

Data will be made available on request.

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