The Impact of Drugs on Hydrogen Sulfide Homeostasis in Mammals

Mammalian cells and tissues have the capacity to generate hydrogen sulfide gas (H2S) via catabolic routes involving cysteine metabolism. H2S acts on cell signaling cascades that are necessary in many biochemical and physiological roles important in the heart, brain, liver, kidney, urogenital tract, and cardiovascular and immune systems of mammals. Diminished levels of this molecule are observed in several pathophysiological conditions including heart disease, diabetes, obesity, and immune function. Interestingly, in the last two decades, it has become apparent that some commonly prescribed pharmacological drugs can impact the expression and activities of enzymes responsible for hydrogen sulfide production in cells and tissues. Therefore, the current review provides an overview of the studies that catalogue key drugs and their impact on hydrogen sulfide production in mammals.


Introduction
Understanding the physiological roles of gaseous mediators such as hydrogen sulfide (H 2 S) has been the focus of the past two decades of active research (for reviews, see [1][2][3][4]). It is now known that animals, plants, fungi, and bacteria naturally produce H 2 S and use this molecule in a range of biochemical and physiological processes [5]. In mammalian systems, H 2 S acts in cells and tissues and plays roles in mitochondrial function and homeostasis [6], cryoprotection [7], cellular antioxidant function [8], inflammation and inflammatory signaling [9], and tissue repair and wound healing [10]. Other research has shown important functions in the cardiovascular system [11,12]; the brain [13], kidney [14], and bladder [15]; the digestive [16] and urogenital tracts [17]; the respiratory and reproductive systems, such as erectile function [18][19][20]; obesity [21]; longevity and aging-related diseases [22]; fundamental cellular processes such as the cell cycle and apoptosis [23,24]; and the induction of signaling cascades such as p53, NF-κB, and Nrf2 [25][26][27] (summarized in Figure 1). H 2 S is a signaling molecule in mammals, and it is therefore important to understand the biotic and abiotic stimuli that can lead to changes in the levels of this molecule in cells and tissues. Interestingly, several commonly prescribed pharmacological drugs have been reported to alter hydrogen sulfide levels in various model systems [28,29], and evidence relating to these molecules is described herein.  [1,30]. C protein is expressed in the brain, liver, kidney, and nervous system [31], and the CSE p tein is expressed in the cardiovascular system and liver, respectively [32]. In addition, H can also be produced in a coupled reaction between 3-mercaptopyruvate sulfurtransfera (3-MST, EC 2.8.1.2) and the enzyme cysteine aminotransferase enzyme (CAT, EC 2.6.1 enzymes located in the mitochondria [33][34][35]. Once produced, H2S orchestrates its effe via the activation of cell signaling pathways, the activation of ion channels, or by prom ing sulfhydration of protein targets. Alternatively, H2S is degraded via oxidative proces to thiosulfate (S2O3 2− ), then sulphate (SO4 2− ). The major enzymes mediating the detoxifi tion of H2S are ethylmalonic encephalopathy protein 1 (ETHE1, EC 1.13.11.18), mitocho drial sulfide-quinone oxidoreductase (SQR, EC 1.8.5.4), thiosulfate sulfurtransferase (T EC 2.8.1.1), and sulfite oxidase (SO, EC 1.8.3.1). First, SQR cysteine persulfide is form Sulfane can further migrate to glutathione to form glutathione persulfide, or to sulfi and then to thiosulfates. Glutathione persulfide is oxidized by ETHE1, thiosulfate is o dized by TST to regenerate sulfites, and sulfites are then oxidized to sulphates by [36,37]. A portion of H2S is also exhaled or scavenged by methemoglobin to gener sulfhemoglobin in the blood [38,39].
In addition to classical enzymatic routes of synthesis, H2S can also be generated thiosulfate and polysulfide species [40,41]. These chemical routes are complex and me ated via cellular thiol exchange reactions with the sulfur amino acids and the redox m ecule glutathione. These chemical routes are partly responsible for the health-promoti properties of some allium and brassica species [42].
In addition to classical enzymatic routes of synthesis, H 2 S can also be generated via thiosulfate and polysulfide species [40,41]. These chemical routes are complex and mediated via cellular thiol exchange reactions with the sulfur amino acids and the redox molecule glutathione. These chemical routes are partly responsible for the health-promoting properties of some allium and brassica species [42].

H 2 S Signaling
H 2 S is the third gaseous mediator found in mammals, akin to nitric oxide (NO) and carbon monoxide (CO) [11]. H 2 S can act as a scavenger of free radicals [8], direct post-transcriptional modification of cellular proteins via S-sulfhydration [43,44], activate ATP-sensitive potassium channels (KATP) [45] and transient receptor potential (TRP) channels [15], or function as a signaling molecule in numerous pathways.

Diminished Levels of H 2 S Occurs in Diseases
In humans, disease conditions may be exacerbated by changes in endogenous H 2 S production via one of several enzymatic pathways [55]. Most of these diseases have been associated with the loss in capacity of cells and tissues to generate H 2 S; this can result in decreased activity or expression of CSE, CBS, or a combination of the two [56]. Currently, diminished levels of H 2 S have been reported to occur in hypertension [57,58], vascular inflammation in pulmonary hypertension [59], tissue inflammation [60], atherosclerosis [61], diabetes [62,63], coronary heart diseases [64], gastric mucosal injury [65], sexual dysfunction [66], neurodegenerative conditions such as Alzheimer disease [67], dementia [68], and Parkinson's disease [69]. H 2 S levels are also reduced in chronic kidney diseases [70], pulmonary tissues in children [71], and reperfusion injury [72]. Interestingly, replenishment with H 2 S via the use of sodium hydrosulfide (NaHS) or H 2 S donor molecules such as GYY4137 often improves the severity of these conditions in models of disease [73][74][75][76].

The Impacts of Pharmacological Drugs on Hydrogen Sulfide Homeostasis
Changes in the homeostatic levels of H 2 S occur naturally in the cells and tissues of mammals and are heavily influenced by disease status (reviewed in [2]), genetics [55,58], age [77,78], diet [55], and stress [22]. Less widely reported is the influence of conventional pharmacological drugs on H 2 S-generating systems in mammals. Several widely known medications have been reported to impact on the expression and activities of several components of the H 2 S-generating system in mammals [79][80][81]. The current review describes these therapeutic molecules and their effects on H 2 S biosynthetic routes in mammalian systems. See Table 1 for additional details of the studies included in this review [ Table S1]. NSAID: Naproxen (20 mg/kg/day) Animal study: Wistar rats ↑ Gastric mucosal protein expression of CSE was observed when compared to controls, while no effect was noticed on CBS and 3-MST. [89] Aspirin ( ↓ the expression of CBS, CSE and H 2 S production in mesenteries; leading to increase blood pressure. [79] Glucocorticoid: Dexamethasone (1-1000 nmol/L) In vitro: macrophages cells ↓ mRNA and protein levels of CSE and H 2 S production in macrophages [99] Glucocorticoid: Dexamethasone(Animal study: 1 mg/kg, i.p.(In vitro: 1-10 µM) Animal study: male Sprague-Dawley rats.In vitro: human foetal liver cells and rat neutrophils.
Animal study: ↓ H 2 S concentration in both plasma and tissues.In vitro: ↓expression of CSE in both human foetal liver cells and in rat neutrophils. [28] Glucocorticoid: Dexamethasone (10 µM) In vitro: chicken myoblasts ↓ expression of the CSE protein in myoblasts.↓mTOR and p70S6K phosphorylation.↓ protein synthesis.
[100] Worldwide, an estimated 30 million people take NSAIDs per day [105]. Non-steroid anti-inflammatory drugs (NSAIDs) are commonly used to relieve pain and reduce inflammation [106]. These properties are largely due to the impacts of these drugs on the gene transcription and protein synthesis of several inflammatory proteins such as cyclooxygenases (COX, EC 1.14.99.1) [107,108]. Unfortunately, common side effects when these drugs are administered chronically and used long term include gastrointestinal (GI) damage, as well as impacts on the cardiovascular system and kidney [109]. Recently, several researchers have reported that a number of NSAIDs reduce H 2 S production in mammalian systems. The suppression of H 2 S appears to be associated with reduced tissue healing and increased inflammation [110,111]. This realization has led several researchers to develop H 2 S-releasing NSAID hybrid compounds, of which many have reduced side effects by virtue of their capacity to sustain H 2 S levels at physiologically relevant concentrations. The aspects of this research are addressed below.
A plethora of animal models have examined the effects of NSAIDs on H 2 S levels in biological fluids and the impacts on the expression of biosynthetic enzymes in tissues. Recently, it was shown that Ketoprofen administered intragastrically to rats for 7 days caused significant reductions in H 2 S levels in the gastric and intestinal mucosa, leading to GI toxicity. In addition, Ketoprofen treatment altered the intestinal microbiome profile of animals, as well as the expression of the mammalian target of rapamycin (mTOR) and suppressor of the cytokine signaling 3 (SOCS3) pathways. In comparison, in the same model, the H 2 S-releasing donor compound ATB-352) significantly reduced GI tract damage via the preservation of H 2 S levels and suppression of oxidative/inflammatory response pathways in treated animals. It was noted that ATB-352 had an improved safety profile and preserved GI mucosal integrity by inhibiting inflammation in tissues [91].
Several other H 2 S-releasing NSAIDs have now been developed and compared to conventional NSAIDs. Again, many of these novel H 2 S-releasing NSAIDs have clear improvements in safety and efficacy when compared to their parental compounds. Indeed, the H 2 S-releasing analgesic/anti-inflammatory drug (ATB-346) has successfully passed phase two clinical double-blinded trials and is reported to have reduced GI toxicity [112]. Importantly, ATB-346 increased plasma H 2 S levels, which may have contributed to the cytoprotective effects. The impacts of other NSAIDs on H 2 S biosynthetic systems have also come under the spotlight over the last two decades. Magierowski et al. [89] reported that in Wistar rats pre-treated with Naproxen, a significant increased gastric mucosal protein expression of CSE was observed when compared to controls, while no effect was noticed on CBS and 3-MST [89]. The elevations in CSE were proposed to be a defensive mechanism of gastric tissues to the gastric damaging effect induced by Naproxen treatment. Naproxen significantly reduced gastric blood flow compared to controls, while pre-treatment with an H 2 S-releasing Naproxen derivative (ATB-346), or naproxen combined with NaHS, were seen to significantly increase gastric blood flow in rats when compared to Naproxentreated rats. ATB-346 lowered plasma concentrations of IL-6 and TNF-α as compared with Naproxen-treated group. ATB-346 had a protective effect, which could be due to H 2 S formation, leading to activation of the Nrf-2/HO-1 pathway.
Other research has indicated that the gastrointestinal expression of CSE is reduced in mice treated with Aspirin (ASP), Indomethacin, Diclofenac, or Ketoprofen [81]. These researchers have indicated that reduced CSE expression in the GI tract enhances the susceptibility of FXR −/− mice to damages caused by ASP and NSAIDs. FXR is a bile acid receptor required to maintain GI integrity. In the small intestine, FXR maintains the expression of homeostasis pathways such as fibroblast growth factor 19 [113]. Fiorucci et al. [82] reported that NSAIDs, such as Indomethacin and Ketoprofen, reduced gastric H 2 S generation and CSE expression/activity in the gastric mucosa of male Wistar rats [82]. The reduction in H 2 S formation was seen to exacerbate mucosal damage; however, the treatment of rats with NaHS significantly alleviated the gastric injuries induced by these drugs. This finding demonstrates that H 2 S is an important component in maintaining GI integrity mediated by the KATP channel. Other researchers have shown that Diclofenac reduced serum H 2 S concentrations and the expression of CSE and CBS in the stomach of male Wistar rats, and that the novel H 2 S-releasing Diclofenac derivative ATB-337 reduced gastrointestinal-injury in animals [84]. This protective observation accounted for H 2 S production from this molecule, which inhibited the expression of TNF-α and the adherence of leukocyte to the vascular endothelium of mesenteric. Again, this finding points to an important role of H 2 S in the GI tract.
By far, the most widely studied drug known to impact mammalian H 2 S production is the COX-2 inhibitor, Aspirin (ASP). Yang et al. [90] reported that intragastrical administration of ASP to male Kunming mice significantly reduced H 2 S production in the gastric mucosa and caused gastric mucosal injury and lesion formation. Furthermore, in ASPdamaged tissues, the infiltration of inflammatory cells and resultant production of IL-6 and TNF-α, as well as oxidative markers, viz., myeloperoxidase (MPO) induction and GSH depletion, were reported. ASP significantly inhibited H 2 S generation and CSE expression in the gastric mucosa. Interestingly, the negative effects observed for ASP were mitigated using a pH-controlled H 2 S donor, JK-1, to replenish endogenous H 2 S levels. JK-1 reduced ASP-induced gastric lesion formation by lowering the levels of IL-6 and TNF-α production, by inhibiting oxidative stress and by inducing tissue GSH concentrations in the mouse gastric mucosa. Similarly, in mice administered with ASP, CBS and CSE mRNA levels were significantly reduced in gastric tissues by as much as 60-70%, and these reductions correlated with the severity of gastric injury [87]. Moreover, replenishment of H 2 S tissue levels using Na 2 S preserved GI integrity, prevented ASP mediated tissue damage, and was accompanied with the activity of GPBAR1, a receptor expressed in the GI that has a role in maintaining GI barrier integrity. In female albino Swiss mice, ASP significantly reduced H 2 S levels in the liver of animals compared to the control (1.52 to 1.15 nmol/g wet weight, control vs. ASP; p < 0.001) [85]. In male Wistar rats, ASP (125 mg/kg/ig) was reported to cause reductions in the CSE protein expression and H 2 S production of treated animals [88]. Interestingly, this study reported a significant elevation in CBS protein expression in gastric mucosal tissues. The upregulation of CBS can be explained as a compensatory mechanism in the GI that likely results from diminished H 2 S levels. Furthermore, pre-treatment with NaHS prevented damage induced by ASP by increasing the gastric bloodflow in treated animals [88].
In male Sprague-Dawley rats treated with ASP, a significant reduction in H 2 S concentration in gastric tissues was reported but no effects on plasma H 2 S concentrations were noted [86]. Researchers have indicated that as H 2 S-releasing derivatives of ASP, ACS14 protects against gastric damage induced by ASP. These protective effects occur by virtue of its H 2 S-releasing capacity. H 2 S production by this molecule was found to inhibit oxidative stress, viz., inhibition of gastric malondialdehyde (MDA) levels and to increase tissue GSH levels. In contrast, some studies have shown that ASP can increase H 2 S concentrations in the liver and brains of mice; however, it is unclear why this may be the case [83].

Paracetamol
Paracetamol (N-acetyl-para-aminophenol; APAP) is a commonly used analgesic among the general population worldwide [114]. It is widely used among pediatric and adult populations for the treatment of pain and fever [115]. Paracetamol is a well-tolerated and remarkedly safe drug if used within the recommended dose, viz., 4 g/d [116]. Indeed, adults typically take from 0.5 to 1 g of oral Paracetamol every 4-6 h, up to a maximum of 4 g in a 24 h period [117]. Beyond this, the number of cases of liver damage caused by Paracetamol is steadily increasing every year worldwide [118]. Studies have shown that most of the toxic cases linked to Paracetamol, including acute liver failure, were due to chronic poisoning [119]. However, although physiological changes with the aging process can impact Paracetamol pharmacokinetics, no specific dose recommendations were advised for older people [120]. The elderly may be at greater risk of Paracetamol toxicity. Mitchell et al. [121] reported on the reductions in serum Paracetamol concentrations among old participants (70 years and above) compared to younger people (18-55 years) after 5 days of 3-4 g/d of oral Paracetamol consumption (p < 0.004). This may indicate reductions in Paracetamol clearance with aging [122].
Research on Paracetamol has spanned several decades since its introduction to clinical use in 1950 [123]. This molecule is known for its analgesic and antipyretic properties, which are similar to NSAIDs. There is little evidence of Paracetamol having any anti-inflammatory properties [124].
Recent evidence has emerged showing that APAP can impact H 2 S levels in mammals, although the data are quite sparse. In studies assessing the role of JNK/MAPK signaling in APAP-induced acute liver failure, Li et al. [93] reported that in male C57BL/6J mice, APAP, reduced the protein expression of both CBS and CSE in liver tissues. Moreover, pre-treatment with the H 2 S donor NaHS preserved tissue CSE and CBS expression levels in animals and reduced APAP toxicity. Molecular interrogation showed that H 2 S inhibited APAP-induced JNK activation in vivo. Moreover, in human hepatocyte cell lines (7702) treated with H 2 S, the activation of caspase 3, Bax, and Bcl-2 expressions were inhibited, and the phosphorylation status of JNK was reduced in hepatocytes. Combined, the protective effects of H 2 S were linked to the inhibition of apoptosis via suppression of the JNK/MAPK signaling pathway.
Similarly, in Wistar rats treated with APAP, CBS and glutathione synthase enzyme expression were reduced in the livers of treated animals, and this corresponded with diminished liver GSH levels and hepatocellular damage (p < 0.05) [80]. Whereas H 2 S levels were not measured in this study, it is interesting to speculate that the reduced capacity of CBS to generate H 2 S could have added to the damage caused by APAP treatment. It is now widely known that H 2 S is protective in models of APAP toxicity. Indeed, H 2 S can stimulate cysteine transport and increase hemeoxygenase (HO-1) expression and GSH concentration in tissues [125,126]. These effects prevent elevations in serum ALT, reduce the levels of hepatic MDA, and preserve GSH. Interestingly, co-treatment with N-acetylcysteine (NAC), a H 2 S-releasing molecule [127], significantly reduces MDA levels, increases tissue GSH levels and the expression of CBS in the livers of APAP treated animals. Furthermore, several studies have now shown that H 2 S supplementation in combination with APAP prevents toxicity in animals. Treatment with NaHS prevented APAP-induced kidney injury and preserved glomerular structures and function in Wistar albino rats administered APAP [128]. The protective effects were linked to the reduction in acute kidney injury (AKI) makers such as KIM-1 (Kidney Injury Molecule-1) and NGAL (neutrophil gelatinase-associated lipocalin). Other notable changes included reductions in the levels of TNF-α, TGFβ, and apoptosis in tissues. This work suggests that in this model, H 2 S had an anti-inflammatory effect in the kidney. Similarly, Morsy et al. [129] showed that administration of NaHS to Swiss mice treated with APAP had a significant reduction in the serum ALT levels and TNF-α expression in liver, in addition to reductions in hepatic MDA and nitric oxide (NO) [129]. This research shows that H 2 S had an anti-inflammatory effect in animals, leading to reductions in APAP-induced hepatoxicity. Other studies using CSE knockout animals have also pointed to roles of H 2 S in APAP-mediated toxicity in animals. The administration of NaHS was reported to suppress markers of liver damage in CSE KO mice, viz., ALT, AST, lactate dehydrogenase (LDH), and MDA in animals administered APAP. Researchers concluded that H 2 S reduced hepatotoxicity directly through the scavenging of N-acetyl-p-benzoquinoneimine (NAPQI) [130]. Other studies have shown that APAP reduces H 2 S levels in the brain of animals. Wiliński et al. [92] showed that in CBA-strain female mice, mice treated with 30 mg/kg/d or 100 mg/kg/d of APAP for five days had significantly reduced brain H 2 S concentration compared to a control group (1.47 ± 0.02 µg/g vs. 0.80 ± 0.02 µg/g vs. 1.05 ± 0.02 µg/g; p < 0.01: control vs. 30 mg vs. 100 mg) [92]. Collectively, of the available evidence, APAP appears to reduce the expression of H 2 S biosynthetic tissues in mammals, and the use of H 2 S donor molecules seems to prevent hepatotoxicity. Clearly, additional studies are warranted to further explore the molecular mechanisms of action of H 2 S in APAP-mediated toxicity.

Anticancer Drugs
Few studies have explored the impact of chemotherapeutic drugs on H 2 S biosynthetic pathways in mammalian systems. What is currently known points to an interesting picture of H 2 S dysregulation that deserves further research. Cisplatin (cis-Diamminedichloroplatinum) is one of the most potent anticancer drugs for the treatment of solid cancers such as testicular, ovarian, head and neck, bladder, lung, cervical cancer, melanoma, and lymphoma, and is used as a first-line medicine in tumor treatment [131,132]. This molecule exerts its anticancer activity via several mechanisms, primarily by causing DNA lesions by interacting with purine bases [133]. In turn, the formation of Cisplatin DNA interactions drives the induction of signal transduction cascades linked to apoptosis in cells and tissues [134]. In patients, Cisplatin use is linked to the development of renal damage in 30% of patients [135]. It is only recently that studies have shown a direct link between Cisplatin-mediated toxicity and H 2 S. In male C57BL/6 mice exposed to Cisplatin, this drug promoted a decrease in expression for both CBS and CSE in the kidneys of treated animals (p < 0.01). Moreover, pre-treatment with GYY4137, a slow-release H 2 S donor molecule, aggravated renal injury induction through an elevation in the inflammatory response and the promotion of apoptosis [95]. In male Wistar rats, Cisplatin induced H 2 S formation in tissues and increased the expression of CSE in renal tissues of animals [94]. Moreover, the inhibition of CSE using DL-propargylglycine (PAG) prevented renal damage induced by Cisplatin. The reduction in renal damage correlated with diminished levels of apoptotic cells and the production of the proinflammatory molecule TNF-α levels in renal tissues. In contrast, Cao et al. [29] indicated that Cisplatin treatment in vitro significantly reduced the expression level of CSE in renal proximal tubular cells, leading to an impairment of H 2 S production in renal tissues, which likely contributed to renal toxicity. Moreover, NaHS treatment alleviated renal dysfunction. The molecular mechanism for these protective effects contributed to the reduction in ROS production.

Statins
Statins are prescribed for the management of high cholesterol levels in individuals due to their effects at reducing low-density lipoprotein (LDL) cholesterol levels in the blood [136,137]. Statins function by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which enhances the rate-limiting step in the biosynthesis of cholesterol [138]. To date, approximately 145.8 million people use statins across 83 countries [139]. In the UK, around one in three people aged 45 years or over take statins [140]. Clear benefits have been reported for the use of these drugs to reduce the relative risk of cardiovascular events in the general population (reviewed by Sirtori [141]. However, several reported side effects are known, including muscle toxicity and liver enzyme effects [142]. The impacts of statins on H 2 S-generating systems are complex, with research showing both inhibitory as well as stimulatory impacts in animal and cell culture models. In murine Raw 264.7 macrophages treated with Fluvastatin or Atorvastatin, the mRNA and protein expression levels of CSE were increased in concentration-and time-dependent manners [98]. These changes correlated with parallel elevations in H 2 S in stimulated macrophages. Importantly, the inhibition of CSE with using dl-propargylglycine (PAG) or siRNA markedly reduced the H 2 S production in Fluvastatin-treated cells. Moreover, the PI3K inhibitor LY294002 and Akt inhibitor perifosine were able to reverse the increases of CSE mRNA and H 2 S production in Fluvastatin-stimulated macrophages. These observations confirm the role of P13 kinases and the AKT signaling pathway in H 2 S production. Other research has shown that in female CBA mice, Atorvastatin treatment (5 mg/kg/day) and (20 mg/kg/day) for five days decreased the level of H 2 S in the liver tissue of animals (3.45 ± 0.03 vs. 3.27 ± 0.02 vs. 3.31 ± 0.02; p < 0.01: control vs. 5 mg vs. 20 mg), but increased the levels of H 2 S in the kidney, brain, and heart tissues of animals [96]. This pattern indicates the complexity of H 2 S biosynthetic systems in different organs systems and their sensitivity to drugs. Similarly, Wójcicka et al. [97] showed that Pravastatin and Atorvastatin increased H 2 S production in the liver of animals by 51.7% and 70.7% in male Wistar rats, respectively [97]. Interestingly, the observed increase in H 2 S was associated with reductions in the mitochondrial oxidation of this gas. Furthermore, several novel H 2 S sensitive probes have been developed to explore the dynamics of H 2 S production in cells following statin treatment. For example, Zhang et al. [143] developed a near-infrared (NIR) fluorescence emission probe sensitive to H 2 S with high selectivity and sensitivity [143]. The developed probe, NIRDCM-H 2 S, was composed of a dicyanomethylene-4H-pyran (DCM) chromophore as the NIR fluorescence reporter and a pyridine-disulfide-propionate group as the responsive site toward H 2 S. This probe could be applied to the monitoring of endogenous production of H 2 S in raw264.7 macrophages in response to Fluvastatin treatment. Fluvastatin was found to promote the activity of CSE and the generation of H 2 S in murine macrophages. Other probes included an encapsulated semi-cyanine-BODIPY hybrid dye (BODInD-Cl) and its complementary energy donor (BODIPY1) into the hydrophobic interior of an amphiphilic copolymer (mPEG-DSPE). The developed radiometric fluorescent H 2 S nanoprobe was used in the trapping of endogenous H 2 S generation in raw264.7 macrophages upon stimulation with Fluvastatin. In parallel to previous reports, Fluvastatin induced CSE upregulation, leading to increased endogenous H 2 S generation in Fluvastatin-stimulated macrophages. These increases correlated with the activation of the Akt signaling pathway [144].

Glucocorticoid
Glucocorticoids are a group of medications that has anti-inflammatory and immunosuppressive effects. This class of drug works via interactions with the glucocorticoid receptor in various cell types [145]. Glucocorticoids have been used widely since the 1950s to treat many diseases such as autoimmune disorders, asthma, inflammatory bowel diseases, and rheumatoid diseases. However, this medication group has adverse effects associated with long-term use, such as osteoporosis and muscle atrophy [146,147].
In an in vivo study, the expressions of CBS and CSE were downregulated following 8-day treatment with Dexamethasone, as well as the production of H 2 S in mesenteric carotid arteries in male Wistar rats (p < 0.05) compared to vehicle group. The observed reduction in H 2 S was accounted for the impairment of CBS and CSE expression, which led to an increase in the risk of hypertension [79]. This finding suggests that the inhibition of H 2 S production induced by Dexamethasone contributes to hypertension development in rats. Tai et al. [148] found that N-acetylcysteine (NAC), a reported H 2 S donor, could inhibited Dexamethasone-induced hypertension in adult male rats. The protective effect of NAC appeared to be linked to its capacity to increase plasma GSH, decrease oxidative stress, and increase protein levels of 3-MST in renal tissues of rats.
Similarly, evidence from several in vitro studies have shown Dexamethasone can suppress LPS-induced CSE expression and the production of H 2 S and NO in macrophage cells. Indeed, l-arginine increased, whereas N(G)-nitro-l-arginine methyl ester (l-NAME) decreased LPS-induced CSE expression and H 2 S production [99]. Moreover, the results suggest that H 2 S may exert anti-inflammatory effects by inhibiting NO production and that Dexamethasone can inhibit CSE expression and H 2 S production. Li et al. [28] showed that Dexamethasone administered either 1 h before or 1 h after LPS administration in rats inhibited the rise in plasma cytokine (interleukin [IL]-1β, tumor necrosis factor [TNF]α), nitrate/nitrite (NO×), soluble intercellular adhesion molecule-1 (sICAM-1) concentrations, and lung/liver myeloperoxidase activity in animals, which is indicative of an anti-inflammatory effect [28]. However, it was also noted that Dexamethasone treatment reduced H 2 S concentrations in both plasma and liver tissues of male Sprague-Dawley rats. In human fetal liver cells and in isolated rat neutrophils, Dexamethasone also reduced the LPS-induced upregulation of CSE in cells. In chicken myoblasts treated with Dexamethasone, Dexamethasone was reported to inhibit protein synthesis, downregulate mTOR and p70S6K phosphorylation, and suppress the expression of the CSE protein in myoblasts. Moreover, L-cysteine and NaHS could abolish the inhibitory effects of Dexamethasone [100]. The abovementioned study indicates that H 2 S plays an important role in the skeletal muscle response to Dexamethasone by suppressing the protein synthesis induced by Dexamethasone. In murine calvaria-derived osteoblastic MC3T3-E1 cells, Dexamethasone downregulated CSE and CBS in osteoblastic cells. Interestingly, NaHS pre-treatment reduced Dexamethasone-induced apoptosis and LDH leakage. In this instance, H 2 S was reported to significantly activate AMPK signaling via the inhibition of ROS production and ATP depletion [101]. Other studies have reported on how Dexamethasone-induced bone loss is associated with decreased levels of serum H 2 S. Reductions in H 2 S were linked to losses in two key H 2 S-generating enzymes in the bone marrow, namely CBS and CSE. Interestingly, treatment with the H 2 S donor GYY4137 prevented a Dexamethasone-induced loss in bone formation. Mechanistically, GYY4137 promoted osteoblastogenesis by activating Wnt signaling through the increased production of the Wnt ligands. In comparison, blockage of the Wnt/β-catenin signaling pathway significantly alleviated the effect of H 2 S on osteoblasts. Collectively, findings from this study have inspired the development of novel H 2 S releasing steroidal therapeutics. Indeed, Corvino et al. [149] recently described the design, synthesis, and pharmacological assessment of novel glucocorticoids-H 2 S donor molecules. These compounds were based on two corticosteroids, namely Prednisone and Dexamethasone, that contained a H 2 S-donating moiety, consisting of 4-hydroxy-thiobenzamide (TBZ) and 5-(p-hydroxyphenyl)-1,2-dithione-3-thione (ADT-OH) residues. Glucocorticoids-H 2 S donors demonstrated prolonged chemical stability at both acidic and physiological pH levels, as well as the capacity to effectively inhibit mast cell degranulation. When assessed in vivo, H 2 S-releasing molecules could reduce the peribronchiolar density of collagen as well as the thickness of the smooth muscle actin better than prednisone.

Phosphodiesterase (PDEs) Inhibitors
Phosphodiesterase (PDE) is a group of enzymes that breaks down the cyclic nucleotides of cAMP and cGMP. Therefore, PDE inhibitors increase intracellular cAMP and/or cGMP, causing muscle relaxation (vasodilatory effect) [103]. PDE inhibitors are a class of medications that have 11 types (PDE1-PDE11) prescribed for the management of conditions such as lower urinary tract symptoms (LUTS), chronic obstructive pulmonary disease (COPD), erectile dysfunction, and atopic dermatitis [102,150]. Headache and gastrointestinal problems such as nausea are commonly reported side effects [151].
Recently, an association between H 2 S levels and PDE inhibitor usage was reported. In an in vitro study, Fusco et al. [102] reported that Sildenafil, a PDE-5, relaxed the human bladder tissue in concentration-and time-dependent manners. However, pre-incubation with DL-propargylglycine (PAG) and aminooxyacetic acid (AOAA), CBS and CSE inhibitors, significantly reduced Sildenafil-induced relaxation (p < 0.001). Moreover, treatment with Sildenafil was seen to significantly increase H 2 S production in a concentration-and timedependent manners in the human bladder dome. In contrast, CBS and CSE inhibition using PAG and AOAA was observed to significantly decrease Sildenafil-induced H 2 S production. This result suggests the involvement of H 2 S in Sildenafil-induced bladder muscle dilations. In pig and human bladder neck, Ribeiro et al. [103] observed that Rolipram, a PDE-4 type inhibitor, had concentration-dependent relaxation effects in tissues. However, the relaxation effect of Rolipram was reduced by blunting H 2 S production with the CSE inhibitor (PAG). This observation suggests the relaxation response is generated partly via H 2 S production in bladder tissues. Similarly, Agis-Torres et al. [104] reported that Rofumilast, a PDE-4, significantly elevated H 2 S production in pig bladder neck samples. When considered together, these findings reinforce a possible role for the H 2 S pathway in the action mechanism of PDE inhibitors in muscle relaxations to manage various diseases.

Conclusions
The impacts of common medications on cellular and tissue H 2 S formation are becoming more widely reported. Many of these animal and cell studies have indicated that several classes of drugs impact H 2 S homeostatic systems by decreasing the expression of enzymes needed for H 2 S biosynthesis such as CSE and CBS. Other studies have shown that some therapeutics reduce the mitochondrial oxidation of H 2 S in the mitochondria. The available evidence shows that two opposing systems are affected in mammalian systems, both critical in maintaining levels of H 2 S in vivo. Less widely known is whether the changes in H 2 S levels precipitate some of the known side effects of many commonly used drugs or if the replenishment of H 2 S using H 2 S donor molecules such as GYY4137 can mitigate potential side effects. In addition, there is a lack of information relating to whether changes in H 2 S production following drug treatment differ between organ and tissue systems or if compensatory mechanisms are induced to address the loss in function of individual H 2 S biosynthetic enzymes. Indeed, Atorvastatin elevates H 2 S formation in periaortic adipose tissue and liver of animals; however, it has little effect in aortic tissues. Importantly, as far as the authors are aware, there have been no intervention studies to study how drugs such as Paracetamol or NSAIDs impact the circulatory levels of H 2 S in humans. Clearly, further research is needed in this area to help clarify the significance of H 2 S dysregulation in tissues caused by common drugs.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/antiox12040908/s1, Table S1: Studies utilizing animal and/or cell-culture models to explore the impacts of common pharmacological drugs on H 2 S homeostasis.
Author Contributions: All authors have equally contributed to this research, and all authors were responsible for preparing and writing the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research was supported by the Saudi Arabian Government and the Jazan University, Saudi Arabia. The Jazan University had no role in the design, analysis, or writing of this article.