Potential exhaled breath biomarkers identified in chlorine-exposed mice

Abstract

Exhaled breath (EB) contains various volatile organic compounds (VOCs) that can indicate specific biological or pathological processes in the body. Analytical techniques like gas chromatography–mass spectrometry (GC–MS) can be used to detect and measure these exhaled biomarkers. In this study, the objective was to develop a non-invasive method of EB sampling in animals that were awake, as well as to analyze EB for volatile biomarkers specific for chlorine exposure and/or diagnostic biomarkers for chlorine-induced acute lung injury (ALI). To achieve this, a custom-made sampling device was used to collect EB samples from 19 female Balb/c mice. EB was sampled both pre-exposure (serving as internal control) and 30 min after exposure to chlorine. EB was collected on thermal desorption tubes and subsequently analyzed for VOCs by GC–MS. The following day, the extent of airway injury was assessed in the animals by examining neutrophils in the bronchoalveolar lavage fluid. VOC analysis revealed alterations in the EB biomarker pattern post-chlorine exposure, with eight biomarkers displaying increased levels and six exhibiting decreased levels following exposure. Four chlorinated compounds: trichloromethane, chloroacetone, 1,1-dichloroacetone and dichloroacetonitrile, were increased in chlorine-exposed mice, suggesting their specificity as chlorine EB biomarkers. Furthermore, chlorine-exposed mice displayed a neutrophilic inflammatory response and body weight loss 24 h following exposure. In conclusion, all animals developed an airway inflammation characterized by neutrophil infiltration and a specific EB pattern that could be extracted after chlorine exposure. Monitoring EB samples can readily and non-invasively provide valuable information on biomarkers for diagnosis of chlorine-induced ALI, confirming chlorine exposures.

Introduction

Exhaled breath (EB) biomarkers refer to biomolecules or other substances that can be detected and measured in a person’s breath, providing insights into specific biological or pathological processes occurring in the body. The analysis of EB, known as breathomics or breath analysis, falls under the field of metabolomics and involves studying the composition of these biomarkers (1–3). The lungs play a crucial role in efficiently exchanging gases, including volatile metabolites and biomarkers between the lung and blood circulation. Even in the early stages of a disease, these biomarkers can be generated and detected (2, 4). By continuously sampling and pre-concentrating exhaled compounds, it becomes possible to analyze an EB sample representative of the entire circulating blood volume. Volatile organic compounds (VOCs) produced in various peripheral tissues are transported to the breath through the pulmonary circulation and subsequent alveolar–capillary gas exchange. These biomarkers can serve both diagnostic purposes and as indicators of exposure to certain substances (1, 3, 5–7). Some of the VOCs have shown promise as potential biomarkers for specific lung diseases like lung cancer (8), asthma (9) and acute respiratory distress syndrome (10). Furthermore, EB can also contain biomarkers other than VOCs, including gases such as carbon monoxide, nitric oxide and hydrogen peroxide, among others (11).

Chlorine is a highly toxic substance that can cause severe respiratory complications within a short period after inhalation exposure. It possesses strong oxidizing properties and intermediate water solubility and can react with the mucus membranes of the airways, potentially leading to acute lung injury (ALI) or even death, depending on the concentration (12, 13). Furthermore, exposure to chlorine can induce the formation of reactive oxygen species, which causes oxidative damage to epithelial cells and other cellular lung components (14, 15). In response to chlorine-induced injuries, the body initiates a repair process through systemic immune activation of complex pathways including signaling molecules, growth factors and extracellular matrix components, which may contribute to a distinct EB pattern that may be specific to chlorine exposure (12, 16). Nevertheless, when exposed to elevated levels of chlorine, this repair process becomes ineffective, potentially resulting in injuries that exhibit characteristics similar to fibrosis and bronchiolitis obliterans (17–20).

Chlorine plays a significant role as an industrial chemical, finding applications in various domains, including chemical synthesis, water purification and bleaching (21). Handling of toxic compressed gases is extensive in industrialized societies, which results in increased risks of unpredictable chemical incidents and antagonistic events (22). The toxic properties of chlorine have led to its deployment as a chemical weapon in armed conflicts, as recent documentation by the Organization for the Prohibition of Chemical Weapons (OPCW) reveals in the conflict within the Syrian Arab Republic (23, 24). The dual use of chlorine in conflict areas emphasizes an urgent need for a robust verification regime to uphold the Chemical Weapons Convention (CWC). Incorporating nasal lavage fluid (NLF) sampling as a non-invasive analytical method to confirm chlorine exposure has shown potential. However, the use of NLF has not been proven in humans exposed to chlorine; it has only been demonstrated in vitro so far (25). There is a requirement to employ sampling techniques with minimal risk of side effects, particularly when compared to invasive methods such as lung biopsies, blood sampling or bronchoalveolar lavage fluid (BALF) when verifying chlorine exposure (25–31). Comparing non-invasive methods, measuring EB is significantly less complex than NLF sampling. Unlike NLF, EB does not require any solvent during sampling or cold storage until analysis. Breathomics presents various advantages, including lower risk of side effects, real-time measurements, ease of sample collection and the ready availability of necessary equipment and technical expertise. These attributes make breath analysis feasible even in conflict zones. Utilizing breathomics as a method for identifying and confirming specific biomarkers associated with chlorine exposure holds great potential for verifying the use of chlorine as a chemical weapon. This approach can provide evidence of individuals being exposed.

In research involving small animals, specialized equipment such as metabolic chambers or breath collection systems can be used to collect breath samples (32–36). For example, analyzing the EB of experimental mouse models of chemical-induced lung injury can provide valuable insights into various aspects, including toxic effects and potential risks to humans. In this study, we have developed a custom-made device to collect EB from mice, allowing us to conduct a comprehensive analysis of the correlation between VOC patterns and chlorine exposure. Our objective was to characterize the EB from chlorine-exposed mice for specific VOCs or VOC patterns that could serve as biomarkers for chlorine exposure or chlorine-induced lung injuries and ultimately be utilized for verification of human exposure.

Experimental

Animals and chlorine exposure

This study was conducted in female Balb/c mice with an age of 10 weeks (average weight 18.8 ± 0.3 g, n = 19) obtained from Envigo RMS B.V. in the Netherlands. The mice were housed in groups of six, and their cages were enriched with pipes and absorbent bedding materials to facilitate nest building. They were maintained under controlled conditions of a 12-h daylight cycle, a temperature of 22°C and 50–60% humidity. The mice had unlimited access to water and food (ssniff, Scanbur, Sweden). The care of the animals and experimental protocols were approved by the regional ethics committee for animal experiments in Umeå, Sweden according to Directive 2010/64/EU.

For the exposure trials, the mice were individually placed in nose-only inhalation exposure tubes (IET 100 B, EMMS, UK), which were connected to an inhalation tower (Battelle). The inhalation tower ensured that all mice received equal and simultaneous exposure to chlorine (supplied by Air Liquide, Germany). The chlorine gas, provided in gas cylinders, consisted of 1 mol% chlorine and 99 mol% nitrogen, was diluted with air to achieve a final exposure concentration of 300 parts per million (ppm), and the mice were exposed at a gas flow rate of 5 L/min for 30 min. The chlorine concentration within the inhalation tower was carefully controlled using a mass flow regulator (LOW-ΔP-FLOW—thermal mass flow meter/regulator, OmniProcess, Sweden) and continuously monitored throughout the exposure period. All experiments involving toxic gas exposures were conducted in a dedicated fume hood. After completion of chlorine exposure, the mice were released back to their cages and allowed to remain there for 30 min until collection of EB VOCs (Figure 1).

Figure 1.

Schematic illustration and a timeline highlighting key points of the experimental design of the (A) exposure (30 min exposure, nose-only, 300 ppm chlorine (Cl₂)) and exhaled breath sampling (30 min), (B) analysis (automated thermal desorption-gas chromatography–mass spectrometry (ATD-GC–MS)) and monitoring of Cl₂-induced effects (pulmonary cells in BALF and recordings of body weight and spleen weight) and (C) a picture of the experimental set-up (photo: Swedish defence research agency).

Collection of exhaled breath VOCs

For collection of baseline VOCs (naïve), the mice were placed in clean inhalation exposure tubes that were positioned on a laboratory bench (Figure 1). The nose end of each exposure tube was equipped with a plastic cassette holder, attached and sealed by a surrounding tubing, allowing an airflow to be directed into a thermal desorption (TD) sampling tube (Steel, Tenax TA 60/80, Markes international, UK) via a Teflon tubing. Calibrated personal air sampling pumps (Gil-Air +, Sensidyne, USA) provided the airflow. For the best achievable isolation of EB, the flow was kept at 40 mL/min to roughly equal the respiratory ventilation of the animal and avoid excessive dilution with ambient air. Each sampling was carried out for 30 min (a sampled volume of 1.20 L) and every sample occasion (three to five animals) was complemented with a simultaneously collected system blank sample (1.20 L) using an empty exposure tube placed next to the animal tubes. For the chlorine-exposed animals, VOC was sampled 30 min post chlorine exposure and the same procedure as described earlier was performed (Figure 1).

VOC collection was made on 19 unexposed (naïve) and 18 chlorine-exposed animals, of which 11 were paired (pre- and post-exposure). In addition, VOC was collected from three exposed and euthanized animals for investigation of volatile markers originating from the body of the mice. The euthanasia was performed 30 min after chlorine exposure and immediately before being placed in the exposure tube before VOC sampling.

VOC analysis

Before sampling, all sample tubes were spiked with 10 µL of internal standard (IS) (480 pg/µL of toluene-d₈ and 2,4-dichloroanaline) in methanol followed by pumping 600 mL of clean air at a flow rate of 200 mL/min to remove methanol. After sampling, the tubes were dried by purging 200 mL of pure N₂. The samples were analyzed employing TD (Unity-Ultra-xr, Markes International, UK) coupled to GC–MS (7890A GC/5975C MSD, Agilent, USA). The TD settings were: primary desorption 300°C, Tenax cold trap 10–250°C (Max rate, >40°C/min) and 2 min hold using an outlet split ratio of 1:6. The GC oven (column: DB 625 UI, 30 m × 0.25 mm i.d. × 0.25 µm film thickness Agilent J&W Scientific, USA) was initially held at 35°C for 2 min, followed by 7°C/min to 240°C and a final hold for 10 min. The mass spectrometer (70 eV) was operated in full-scan acquisition (19–350 m/z). After MS calibration with perfluorotributylamine, the response factor for toluene was determined by analyzing Tenax tubes spiked with toluene at three concentration levels (R² = 0.99).

VOC data collection and data pre-processing

Chromatographic profiles and mass spectra of all samples were evaluated using the Automated Mass Spectral Deconvolution and Identification System (Version 2.73 2017, NIST, USA). A comprehensive target library was generated by combining chromatographic peaks from all samples. Prior to its application in sample processing, any peaks identified as artifacts or originating from the sampling environment were excluded. This resulted in a refined library containing spectra for a total of 67 compounds that were identified by comparing mass spectral profiles using NIST Mass Spectral Library 2.3 (as of 2017). Individual peak areas for each detected compound within a sample was normalized by the IS and then relative EB concentrations (ng/L) were calculated using the response factor for toluene.

VOC data analysis

Multivariate data analysis was performed using SIMCA 17.0 (Sartorius Stedim Biotech, Germany). Prior to data analysis, the concentration data were log-transformed and scaled to unit variance. Orthogonal partial least squares discriminant analysis (OPLS-DA) (37) was used to discriminate between chlorine-exposed and naïve mice. To facilitate the examination of individual responses to chlorine exposure for paired animals, the data were normalized by subtracting the pre-exposure measurement. To extract the important compounds related to chlorine exposure, the model weight values (w*) i.e., variable contribution values for the pre-defined sample class separations, was investigated. Variables with w*> ± 1 STD of all w*-values were extracted as important. To depict the distribution of selected variables between the two classes, box-plots based on log-transformed data were generated using R (version 4.0.4).

Observed health effects 24 h after chlorine exposure

Prior to collecting EB samples, the mice were weighed. The experiments were finished 24 h after chlorine exposure by administering an intraperitoneal over-dose of pentobarbital sodium (>90 mg/kg body weight). To evaluate ALI in the mice, the chlorine-exposed animals were compared to naïve mice by assessing body weight loss at termination. After collecting BALF, the spleen was removed and weighed.

BALF was collected for total and differential cell count analysis at 24 h post chlorine exposure. This involved injecting and withdrawing 2 × 1 mL of ice-cold Hank’s balanced salt solution (HBSS, Sigma-Aldrich) through the endotracheal tube (15 G) of euthanized mice. The injection and withdrawal of HBSS were performed to obtain a total BALF volume of 1.5 mL. Subsequently, the BALF was centrifuged (1,500 rpm, 10 min, 4°C) and the resulting pellet was resuspended in 0.5 mL of phosphate-buffered saline (PBS). The number of leukocytes was counted in a Bürker chamber using trypan blue staining. Additionally, from each animal’s BALF sample, 20,000 cells were fixed on duplicate slides using a Cytospin^® centrifuge (Shandon^© cytospin 3 cyto-centrifuge, cell preparation system, Runcorn, UK). These slides were then stained with May–Grünwald–Giemsa reagents (Merck Millipore, VWR International, Sweden). The slides were blinded, and a differential count of leukocytes, including macrophages, neutrophils, eosinophils and lymphocytes, was performed by counting 300 cells per slide. The statistical data presented are expressed as mean ± standard error of the mean (SEM) and evaluated using an unpaired t-test. A P < 0.05 was considered to be statistically significant. Graphs were generated using GraphPad Prism program (version 9.5, GraphPad Software Inc., San Diego, CA, USA).

Results and discussion

Analysis of exhaled breath VOCs

To meet the objectives of the study, it was necessary to develop a methodology for capturing and analyzing EB from small animals. The developed method ensured the ability to collect and analyze EB samples from the animals, as well as from ambient air samples, with sufficient sensitivity to accurately measure VOCs in the small volumes of breath produced by mice (Figure 1). Samples were collected for 30 min in various conditions: (i) from mice before and 30 min after chlorine exposure in an animal exposure tube, (ii) from euthanized chlorine-exposed animals and (iii) from environmental room air passing through an empty exposure tube (to account for environmental fluctuation in the laboratory). Animals were euthanized to distinguish between VOCs originating from the lungs and those originating from the body or the environment. This ensures an accurate assessment of lung-derived VOCs.

The composition of breath in mice exhibits variations when compared to that of humans. Isoprene, the predominant VOC in human breath, was barely detectable in mouse breath, and acetone, another abundant VOC in human breath, was also found in lower concentrations in mice (38, 39). Figure 2 displays VOC profiles from a mouse (representative of this study) and a typical human breath sample as reported by Philips et al. (39). Some VOCs detected in mice breath included terpenes, a group of compounds predominantly produced by plants, specifically α- and β-pinene. However, even though these terpenes have been detected in human breath as well (39), they were excluded from the target library. This decision was made because their presence could potentially be linked to the wood chips found in the mice’s living environment, and as a result, they were considered contaminants.

Figure 2.

Representative chromatographic profiles of VOCs in exhaled breath from (A) naïve mice and (B) human with a selection of normal breath VOCs highlighted (39).

EB profiles of naïve and exposed mice were investigated by multivariate statistical analysis. The generated OPLS-DA model (CV-ANOVA = 1.2 × 10⁻⁸) showed almost complete discrimination between naïve and chlorine-exposed mice (Figure 3A). Of the VOCs responsible for the classification, based on the previously described w* criteria, eight VOCs were significantly increased in exposed mice while six VOCs were decreased (Table I). It cannot be ruled out that the reduction in the concentration of certain VOCs may be partially attributed to a dilution effect. This effect could result from collecting the same air volume from both naïve and exposed animals, despite differences in breathing patterns, such as decreased tidal volume caused by chlorine exposure. In Figure 4, the distribution of the eight most significant variables, with four decreasing and four increasing after exposure, are shown as boxplots for individuals pre- and post-exposure. Because of their chlorine content, trichloromethane, dichloroacetonitrile, chloroacetone and 1,1-dichloroacetone were of particular interest as chlorine-specific exposure markers. Of these, trichloromethane, also referred to as chloroform, exhibited the highest EB concentrations and showed a consistent increase in all exposed individuals, on average 13-fold. However, trace levels were also present in most unexposed naïve animals. Dichloroacetonitrile, chloroacetone and 1,1-dichloroacetone showed lower EB concentrations than trichloromethane and in some exposed individuals they were not detected. On the other hand, they were never detected in naïve mice, indicating their presence as a specific marker of chlorine exposure. The other compounds within the VOC pattern did not contain chlorine and were all detected in mice both before and after exposure, thus likely serving as effect markers, that is, normal VOCs that exhibit changes, either increases or decreases, because of altered metabolic responses or impairment of lung tissue due to the chlorine exposure. Chloroacetone and 1,1-dichloroacetone are chlorinated forms of acetone and given that acetone is abundant in human breath, it raises the possibility that these markers might serve as indicators of chlorine exposure in humans as well. Trichloromethane and dichloroacetonitrile are also chlorinated forms of typical human breath components, which further strengthen the idea that these findings may have relevance also for humans. Trichloromethane also exhibits a significant increase in EB content following swimming pool visits and showering. Breath samples have identified trace amounts of trichloromethane in breath samples even before swimming (40, 41), which is consistent with the findings in the present study. Additionally, traces of trichloromethane can be detected in the indoor air as reported by Levesque et al. (42). Despite trichloromethane being detected in trace amounts in naïve mice (Figure 4G), we categorize it as a chlorine marker due to significantly higher levels in chlorine-exposed mice (P < 0.001).

Figure 3.

Cross-validated score scatter plot showing the differentiation between naïve (unfilled circles) and chlorine (Cl₂)–exposed (filled circles) mice. (A) Differentiation between all naïve (n = 19) and Cl₂–exposed mice (n = 18). (B) Inter-individual response to Cl₂ exposure from paired mice (n = 11) before (unfilled circles) and after exposure (filled circles).

Table I.

VOCs Identified as Exhaled Breath Biomarkers in Chlorine (Cl₂)-exposed Mice

a

Trace amounts of trichloromethane in naïve mice.

Three types of markers were associated with Cl₂ exposure: Cl₂ exposure marker—exclusive to Cl₂ exposure; effect marker—changed after exposure to Cl₂, in comparison to pre-exposure levels in naïve mice; effect marker from the body—a marker originating from the body that changed in Cl₂–exposed animals that were euthanized. The features are putatively identified with a NIST Match Factor > 900. (Blue: upregulated markers. Red: downregulated markers.)

Figure 4.

Boxplots showing the distribution of the eight most altered VOC variables, which decreased and increased in naïve mice (white boxes, pre-exposure, n = 19) and in mice exposed to chlorine (Cl₂) (grey boxes, post-exposure, n = 18). Unpaired t-test was performed between naïve mice and Cl₂–exposed mice to investigate significant differences between groups. (A) Ethanol (C₂H₆O, P = 0.061), (B) isopropanol (C₃H₈O, P = 0.02), (C) propanol (C₃H₈O, P < 0.0001), (D) 2-butanol (C₄H₁₀O, P < 0.0001), (E) dichloroacetonitrile (C₂HCl₂N, P = 0.001), (F) chloroacetone (C₃H₅ClO, P = 0.012), (G) trichloromethane (CHCl₃, P = 0.001) and (H) 1,1-dichloroacetone (C₃H₄Cl₂O, P = 0.007). The black and the gray oval shapes are potential outliers.

The inter-individual response to chlorine exposure was investigated from paired mice before and after exposure. As shown in Figure 3B, the individual response differs between different mice, for example mouse number 8, 9 and 11 show a larger difference in breath biomarker pattern than mouse number 1 and 4. The difference in response is expected and inter-individual variations in VOC response patterns would also be likely for humans. However, it is important to note that all exposed mice exhaled at least two of the chlorine-containing markers, even though some of them showed a lower response based on the complete VOC pattern. Three of the chlorine-exposed animals were euthanized before collection of VOC and the collected data were used to investigate VOCs related to the passive emissions from the body rather than the EB. The euthanized chlorine-exposed animals were classified as chlorine exposed, using the OPLS-DA model, mostly due to the marked increase of 6-methyl-5-hepten-2-one in the euthanized mice samples compared to naïve and exposed animals. This indicates that 6-methyl-5-hepten-2-one is probably a VOC originated from the body rather than from the EB and is thus categorized as an effect marker from the body (Table I).

Observed health effects after chlorine exposure

Prior to chlorine exposure, naïve animals exhibited no signs of stress or abnormal behavior during 30 min of EB collection. However, after a 30-min chlorine exposure, mice displayed visible signs of distress, including breathing difficulties, slower breathing pace and decreased activity. One day after exposure, examination of BALF indicated the presence of a neutrophilic inflammatory response, along with a decrease in body weight and spleen weight (Figure 5). As previously demonstrated, exposure to chlorine can lead to a reduction in spleen weight (16). The combination of reduced body weight and decreased spleen weight can be regarded as indicative of a severe ALI. The reduction in spleen weight could have multiple causes and one suggested explanation is that it reflects the release of immune cells into circulation to support and protect the damaged lung (16). One day after exposure, the visible symptoms observed 30 min after exposure transitioned to lethargy with much less breathing difficulty.

Figure 5.

Chlorine (Cl₂)-induced health effects in mice on (A) body weight (P = 0.001), (B) spleen weight (P = 0.007) and lung infiltration of inflammatory cells, (C) total count of leukocytes (P = 0.001) and (D) neutrophils (P < 0.0001) in BALF at 24 h after exposure to 300 ppm Cl₂ for 30 min. Values indicate means ± SEM. Significant differences (unpaired t-test) compared to Cl₂–exposed mice are indicated in the graph.

Future perspectives

A panel of VOCs associated with chlorine exposure is shown here in mice (Table I). Since some of these VOCs are chlorinated forms of normal human EB components, similar results would probably be detected in humans. There are some challenges associated with breath analysis in mice, including the relatively small size of the breath samples and potential confounding factors such as diet, environmental conditions and obvious limitations for isolation of exhaled air when compared to humans. Standardization of protocols and further research are necessary to enhance the reliability and reproducibility of breathomics in mice. A limitation is that breath analysis may also offer indications of stress responses and changed respiratory rate in mice, especially after chemical exposure. Human breath analysis faces challenges and limitations due to significant variability in composition, influenced by factors like age, diet, lifestyle and health. Establishing universal baseline profiles is difficult, and this will impact the accuracy of results. Challenges also include external interference (environmental factors), lack of standardized protocols for breath collection, temporal variation such as timing of sample collection in relation to exposure or disease onset (43). There are two primary methods for analyzing EB samples from humans or other mammals: direct measurement of VOCs present in EB samples or obtaining exhaled breath condensate (EBC) by cooling the breath. By cooling the breath, the water vapor and small droplets of lung lining fluid in the breath condense and the liquid sample can be analyzed for a wide range of potential analytes such as proteins, inflammatory markers, metabolites and other relevant compounds. Collecting EBC allows for a more comprehensive analysis of a broader range of biomarkers, as the condensate captures a larger variety of molecules present in the breath (3, 44). As far as we know, there has not been an examination of EBC in humans to confirm exposure to chlorine. However, there is a study involving rats, conducted by Elfsmark et al. in 2018 (14), which suggests that EBC could serve as a suitable sample matrix for detecting isoprostane biomarkers related to chlorine-induced ALI. While recent advancements have occurred, there is currently no definitive biomarker for detection and verification of chlorine exposure (25–31). Exploring EBC analysis in humans may hold promise for identifying exposure to this toxic gas. EB, NLF and EBC offer a less complex alternative to matrices like blood, BALF and biopsies. Despite the challenges of invasive sampling, which can lead to increased patient discomfort and complications, the choice between invasive and non-invasive biomarkers depends on the balance of risks and benefits. Invasive matrices have advantages, as they are less affected by external factors, likely offer better precision and specificity and may provide more accurate quantitative measurements, particularly when assessing concentrations in specific tissues compared to EB and EBC, respectively.

Conclusion

To the best of our knowledge, this is the first study that has used breath analysis to investigate chlorine-induced pulmonary VOC patterns as exposure biomarkers. By analyzing and studying the composition of VOCs, insight into exposure levels and potential health effects of chlorine exposure can be determined. These EB VOCs can also serve as potential biomarkers that can indicate the degree of chlorine-induced lung injury and be an effective non-invasive verification method for chlorine exposure to help confirm the use of chlorine as a chemical weapon and to identify individuals who have been exposed to chlorine. The field of breathomics is still evolving, and more research is needed to fully understand its potential applications and limitations, especially when using animal models. The translation of findings from animal models to human applications requires careful consideration and validation to ensure their relevance and reliability.

Data availability

Data available on request.

Funding

This study was financially supported by the Swedish Ministry of Defence and the National Board of Health and Welfare through the Swedish Center for Disaster Toxicology.

Acknowledgments

Karin Wallgren is gratefully acknowledged for thankful help with the animal experiments.