Chemotherapy is a type of anticancer treatment that uses one or more chemical substances (extracts from natural substances or products of chemical synthesis) that stop the multiplication of cancer cells, either by destroying them or by stopping their division. Chemotherapy is an essential component of the pharmacotherapeutic management of cancer. (Sharifi-Rad et al., 2021b; GBD, 2019 Colorectal Cancer Collaborators, 2022). On the other hand, the resistance of cancer cells towards chemotherapeutic drugs has become prevalent, being associated with unfavourable clinical evolution in cancer patients directly and indirectly (Sharifi-Rad et al., 2021e; Sharma et al., 2022). The progress of new drug research and development may overcome the occurrence of drug resistance (Ali et al., 2022b). It has been also reported that natural products and derivatives with diverse chemical structures and pharmacological effects serve as useful compounds against cancer and drug-resistant cancer (Ali et al., 2022a; Dhyani et al., 2022b; Kitic et al., 2022; Sharifi-Rad et al., 2022a). Mycotoxins are types of toxins produced by a variety of fungal species of crops or stored commodities. Mycotoxins appear as primary or secondary contaminants via the carryover effect in the food chain (Degen, 2017). Due to various biological effects, mycotoxins have come to the attention of scientists in the context of research done to discover and develop new anticancer drugs (Islam, 2017; Islam et al., 2018). Generally, mycotoxins are ubiquitous and unavoidable harmful fungal products and vary significantly in structure and biochemical effects. These toxins cause disease in both animals and humans and are found in almost all types of foods, with a higher prevalence in hot, humid environments (El Khoury et al., 2019). Unfortunately, most of the published data has concerned the major mycotoxins aflatoxins, ochratoxin A, zearalenone, fumonisins and trichothecenes, especially deoxynivalenol (Smith et al., 2016), although, there are aspects of mycotoxin relations with strain improvement strategies and genetic modification for improved detoxifying properties in test systems (Pfliegler et al., 2015).

Alternariol (AOH), a toxic mycotoxins metabolite of Alternaria fungi, is an essential contaminant in cereals and fruits. Alternaria fungi are plant and human pathogens, saprophytes, a strong allergen and exposure has been associated with allergic diseases such as allergic rhinitis, chronic rhinosinusitis and asthma (Grover and Lawrence, 2017; Aichinger et al., 2021). Mycotoxins enter the body through contaminated food, but can also enter the airway or through direct skin contact. In general, mycotoxins are resistant to high temperatures, and many mycotoxins are also resistant to industrial food processing, so to have mycotoxin-free foods, the raw material (wheat, milk, vegetables, meat, etc.) must be analyzed. Because they are resistant to processing, they can also be found in highly processed foods such as bread, breakfast cereals, wine, and beer. Many pharmacological activities, including antifungal (da Cruz Cabral et al., 2019), anti-inflammatory (Kollarova et al., 2018), and anticancer effects have been done (Meena and Samal, 2019). This updated review sketches a current scenario of AOH’s anticancer effect and possible action mechanisms behind it based on database information.

A literature study was conducted up to December 2021 using the following databases: PubMed/MedLine, Science Direct, Web of Science, Scopus, and the American Chemical Society using the next MeSH terms: “Alternariol,” “Alternariol monomethyl ether,” “Alternaria/metabolism,” “Mycotoxins,” “Cell Line,” “Tumor,” “Cell Survival/drug effects,” “Humans,” “Mycotoxins/toxicity,” “Reactive Oxygen Species.” No language restrictions were imposed. Articles were evaluated in detail and summarized information on the dose, concentration, administration route, experimental model, results discussion, conclusion, and the proposed action mechanism.

A recent study showed a significant reduction in AOH when exposed to a temperature of 35°C, and very high temperatures above 100°C significantly affect its stability. Compared to this, its derivative, alternariol monomethyl ether (AME) is much more stable; at high temperatures of 80°C–110°C (Estiarte et al., 2018). AOH suffers several reactions of biotransformation, as has been demonstrated by studies performed in vivo, in rat liver slices, cell culture or purified enzymes. The identified chemical modifications of AOH include hydroxylation (phase I biotransformation), sulfation and glucuronidation (phase II biotransformation), which are executed mainly by cytochrome P450 isoforms (Tran et al., 2020). The principal organ of AOH metabolization is the liver, although other organs like the kidneys, the bladder and components of the gastrointestinal tract have also been involved. Of note is the lack of relevant participation of gut microbiota in the biotransformation of AOH (Lemke et al., 2016). Some enzymes responsible for AOH metabolization are uridine 5′-diphosphate-glucuronosyltransferase, glutathione S-transferase and CYP1A1 (Appel et al., 2021). The last one is responsible for hydroxylation at C-2, C-4, and C-8. Subsequently, 4-hydroxy-AOH is glycosylated to 3-glucoside (58%) and 9-glucoside (5%) in the whole-cell system. The metabolite 9-diglucoside can also be hydrolyzed to 9-glucoside. Some metabolites of AOH, as catechols formed by its hydroxylation, can also be methylated and hydroxylated.

In vivo studies performed in rodents reveal that a high percentage (85%–91%) of AOH given orally is excreted in the faeces, and a low percentage in the urine (>2.6%), with 0.8% urine excretion of alternariol-3-sulphate (Schuchardt et al., 2014). The blood concentration of AOH only reaches 0.06% after 24 h when 2000 mg/kg are administrated orally. However, when doses were applied in triplicate at 0, 24 and 45 h, AOH reached a blood concentration of 0.5 µM after 3 h of administration. The study performed by Schuchardt et al. (2014), could detect 4, 10, 8, and 2 hydroxy metabolites of AOH in urine the following three days after the triple doses. Using polarized human colon adenocarcinoma Caco-2 cells culture as a model of the intestinal barrier, it has been established that between 23% and 26% of the apically applied AOH crosses the cell barrier, founding several metabolites on the basolateral side (Appel et al., 2021). When CaCo-2 cells were cultured with AOH or 9-glucoside alternariol, a similar distribution of derivates were found on the apical and basolateral side. Particularly, after 3 h of apical exposure, 45% of the initial compounds of the supernatant corresponded to 9-glucoside, 15% to 3-glucuronide, 14% to 3-sulphate and 11% to 9-glucuronide. Using AOH unconjugated molecule, there was 8% of the recovered compounds. Specifically, in the cell, the glucuronides and the glucoside also could be detected. The 3-glucoside plant metabolite displays a different distribution in the whole cell system, showing over 90% of metabolites recovered. Also, in the cells, only traces of the same metabolites were detected, including the unmodified AOH. On the other hand, 9-diglucoside has no significant absorption. In summary, the glucuronides and sulfates of AOH showed moderate absorption (20%–70%), meanwhile, the free mycotoxin and the 9-glycoside have higher absorption (Burkhardt et al., 2009). These data reveal that AOH and its metabolites are significatively absorbed by epithelial cells, but the localization of the glycosylation position affects its absorption and metabolization.

The toxicity of AOH has been studied since the 70s, mainly through in vitro models (Pero et al., 1973). However, insufficient in vivo data has prevented the assessment of AOH health risks for different species, including humans (EFSA Panel on Contaminants in the Food Chain et al., 2019). Among the first in vivo data, it is reported that toxins from Alternaria cultures are lethal when injected intraperitoneally at a dose of 100 mg/kg per day in DBA/2 mice (Pero et al., 1973). AOH is one of the 70 mycotoxins present in the Alternaria culture and produces itself, a median lethal dose of 400 mg/kg of body weight when administered in isolation in DBA/2 mice (Woody and Chu, 1992).

Another in vivo study aimed to study the genotoxic potential of AOH administered by oral gavage in NMRI mice (Schuchardt et al., 2014). The oral AOH did not cause an effect on the general health status during 7 days of observation, at doses up to 2000 mg/kg. Of note, the lack of toxicity could be related to the low systemic absorption of AOH, which reached blood levels of 0.5 µM representing less than 0.06% of the administered dose. At this low systemic concentration, AOH was negative for bone marrow micronuclei test and alkaline comet assay in the liver but the assays to investigate local genotoxicity in gastrointestinal tissues failed due to adverse effects of the AOH vehicle (corn oil) (Schuchardt et al., 2014).

A recent study investigated the effect of AOH in early-stage embryonic development through the injection of pregnant mice with AOH for 4 days. The highest dose of 5 mg/kg body weight/day caused injurious effects on embryonic development from the zygote to the blastocyst stage and also embryo degradation. Additionally, AOH also provoked a redox to unbalance in the offspring of mice during early gestation, suggesting that the toxin could act through an epigenetic mechanism (Huang et al., 2021). The reproductive and developmental toxicity of AOH could be related to its ability to act as an estrogen agonist. In this regard, AOH is a diphenolic compound that has some structural similarities to estrogen molecules and acts as a weak estrogen agonist as revealed by reporter assays in H295R cells (Frizzell et al., 2013). However, in other estrogen-responsive cells, like porcine granuloma cells, AOH failed to activate estrogen receptor a (Tiemann et al., 2009). In contrast to the effect on embryonic development in mammals, the injection of AOH into the yolk sac did not cause mortality or teratogenic effect in chicken embryos at doses up to 1 mg per egg (Griffin and Chu, 1983).

There is broader evidence regarding AOH toxicity in vitro models, including studies performed in bacterial strains and mammalian cell lines that show genotoxic activity (Solhaug et al., 2016). In Salmonella strains, AOH induces direct-acting AT base pair mutagenicity (Schrader et al., 2006). Also, its capacity to induce frameshift mutations was probed in Bacillus subtilis and Escherichia coli ND160 (EFSA Panel on Contaminants in the Food Chain et al., 2019). In mammalian cell lines, it has been reported that 1-h exposure to AOH in the range of 5–10 µM causes DNA strand breaks in V79 fibroblasts from Chinese hamsters, HepG2 hepatoma cells and HT-29 colon cells (Pfeiffer et al., 2007). Along with the mutagenic effect, AOH is also responsible for chromosome aberrations that are evident after 48 h of 10 µM exposure to the mycotoxin. Particularly, AOH induces kinetochore-negative micronuclei in Ishikawa and V79 cells, unscheduled DNA synthesis in the primary culture of human amniotic cells and increased mutations at the hypoxanthine phosphoribosyltransferase 1 (HPRT) gene in V79 fibroblasts (Lehmann et al., 2006). Another line of evidence suggests that DNA damage at molecular and chromosome levels is mediated by ROS production induced by AOH (Solhaug et al., 2016). This proposed mechanism is based on cytotoxicity assays of AOH, performed in cell lines including HT29, V79, RAW264.7 and Caco2 (Solhaug et al., 2012; Tiessen et al., 2013; Fernández-Blanco et al., 2014). For example, when Caco2 cells are exposed to AOH in a range of 15–60 µM for 24 h, there is a significant increase in ROS species, lipid peroxidation and a decrease in catalase and superoxide dismutase activities. Despite its oxidative effect, AOH produced a minor reduction in cell viability on Caco2, even at doses of 100 µM for 72 h (Fernández-Blanco et al., 2014).

Tiessen et al. (2013) also reported that AOH and AME induce an oxidative response in HT29 cells, including a transient decrease in glutathione levels, with a short exposure of 1 h. However, this effect did not produce DNA damage probably due to the activation of the redox-sensitive transcriptional response elicited by the transcription of Nrf2 (Tiessen et al., 2013). Another proposed mechanism for AOH genotoxicity is related to the inhibition of DNA topoisomerase I and IIa (Fehr et al., 2009). This enzyme is important to resolve topological constraints during DNA replication and therefore, it is likely that AOH-induced inhibition of topoisomerases could be responsible for the clastogenic effects observed in cell lines.

Therapeutic limitations derive from insufficient knowledge of the pharmacokinetics, solubility, bioavailability, metabolism of alternariol, insufficient understanding of the molecular targets of action at the tumour cellular level, and their regulatory pathways. Although only experimental in vitro pharmacological studies have demonstrated and justified the anticancer effects of alternariol, translational pharmacological studies establishing the effective anticancer dose in humans, as well as clinical studies in humans, are lacking. Also, the development of new nanoformulations of alternariol in which it can be incorporated into different nanocarriers at the target should be the focus of future research. As a result, alternariol cannot be used in anticancer therapy as a first-line treatment, but only as an adjuvant in combination with standard chemotherapeutic treatment.

AOH and its derivatives, such as AME, alternariol-9-methyl ether-3-O-sulphate, alternariol 3,9-dimethyl ether and altertoxin II, exhibit an anticarcinogenic effect through several pathways, with ROS generation leading to the induction of oxidative stress and a cytotoxic effect linked to mitochondrial dysfunction, inflammatory pathway, cell cycle arrest in G0/G1, G2/M and S phases, apoptotic cell death, genotoxic and mutagenic mechanisms, antiproliferative, autophagy, as well as estrogenic and clastogenic mechanisms. To our knowledge, no other studies have explored the anticarcinogenic effect of AOH or its metabolites in animal models or clinical trials. This was corroborated by a search of the literature and also of US and European databases for completed or ongoing clinical trials (www.clinicaltrail.gov, www.clinicaltrailregister.eu). Given these promising results of experimental pharmacological studies, AOH and its derivatives can be considered potential adjunctive chemotherapeutic agents.