Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 1 | 12 ORIGINAL RESEARCH Aflatoxin B1 as an endocrine disruptor among miller flour workers Safia Beshir1, Weam Shaheen1, Amal Saad-Hussein1, Yuosra Saeed2 1Environmental and Occupational Medicine Department, National Research Centre, Giza, Egypt; 2Air Pollution Research Department, National Research Centre, Giza, Egypt. Corresponding author: Weam Shaheen; Address: National Research Centre, Dokki, Giza, Egypt. El-Buhouth St., 12622; Telephone: 01118983587; Email: weamshaheen@gmail.com mailto:weamshaheen@gmail.com Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 2 | 12 Abstract Aim: Aflatoxin В1 has been stated to inhibit the function of different endocrine glands. This study was proposed to clarify the possible effects of aflatoxin B1 as an endocrine disruptor on pituitary gland, thyroid gland and gonads among miller flour workers, and to evaluate its effects on human male sexual function. Methods: A case-control study was conducted in a flour mill in Helwan District Cairo, Egypt in 2019. The study included 42 exposed flour milling male workers from the grinding department which showed the highest level of aspergillus flavus in the air sampling of airborne fungi and 40 non-exposed males. Serumaflatoxin В1/albumin, luteinizing hormone, follicle stimulating hor- mone, testosterone, 17-beta-estradiol, free triiodothyronine, free thyroxin and thyroid stimulating hormone were measured for the studied groups. Results: Sampling of airborne fungi revealed that aspergillus flavus and penicillum were the pre- dominant fungal types in the flour mill. Indoor/Outdoor ratios for aspergillus flavus were ≥ 1 in all the locations indicating presence of indoor sources. Serum Aflatoxin В1/albumin, luteinizing hormone and follicle stimulating, the existence of various types of sexual disorders (decreased libido, impotence and premature ejaculation) were higher while testosterone was lower in the mil- ler flour workers compared to non-exposed. However, there was no significant difference regard- ing 17-beta-estradioland thyroid hormone levels between both studied groups. Conclusion: Aflatoxin B1creates possible human male reproductive health distresses in miller flour workers. Keywords: aflatoxin В1, Egyptianflour workers, LH and FSH, sexual disorders, testosterone, thy- roid hormones. Acknowledgement: The authors are grateful to the National Research Centre for funding this research. Funding: This study was funded by the National Research Centre, Egypt. Conflicts of interest: None declared. Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 3 | 12 Introduction Worldwide, human fertility is deteriorating; a state that cannot be referred only to the in- crease in contraception usage (1). Many en- vironmental factors, industrial and occupa- tional compounds, dietary contaminants, life- style factors and medications were suggested to be other causes to this deterioration (1). Male infertility causes may be pre testicular, testicular and post testicular. The pre testicu- lar and the testicular causes are chiefly endo- crine disorders that originate from the hypo- thalamic-pituitary-gonadal axis that have op- posing effects on spermatogenesis (2). Male reproduction is controlled by the hypo- thalamo-hypophyseal testicular axis: hypo- thalamic gonadotropin releasing hormone, pituitary luteinizing hormone (LH) and folli- cle stimulating hormone (FSH) and the gon- adal steroid, principally, testosterone. It was proved that thyroid hormones have a change- able effect on this axis and thus affect the sex- ual and spermatogenic function of man (3). Effects of thyroid hormones occur through binding to certain thyroid hormone receptors which are extensively spread in the testis (4). Endocrine disrupting chemicals (EDCs) may be of synthetic (pesticides, industrial chemi- cals, bisphenols) or natural origin (mycotox- ins, phytoestrogens). Some mycotoxins can act as probable endocrine disruptors and cause changes in hormone production (5). Endocrine disruptors may simulate the action of sex hormones, affect reproduction (6), cause reproductive anomalies (morphologi- cal and functional gonadal dysfunction, e.g. infertility and decreased libido) and congeni- tal malformations (altered embryonic and foetal intrauterine development) (7). Egypt is one of the countries with high wheat consumption (8). Fungi can produce varied types of mycotoxins under environmental conditions which are favourable to growth. Aflatoxins are naturally occurring mycotox- ins produced by certain fungi, mainly asper- gillus flavus and aspergillus parasiticus. Af- latoxins B1 (AFB1) is one of the main afla- toxin types (9). AFB1 has been stated to inhibit the function of different endocrine glands by disturbing the enzymes and its substrate that are respon- sible for the synthesis of different hormones (10). Aflatoxins have the ability to generate hormonal dysfunction inducing cell toxicity which directly affects reproduction (11). Pre- vious studies stated that AFB1 disturbs the hypothalamo-pituitary testicular axis result- ing in production of malfunctioning sperma- tozoa (12,13). Uriah and his colleagues (14), proved that aflatoxin levels in the blood and semen of infertile Nigerians men were signif- icantly higher than in the fertile men, sug- gesting that aflatoxin might be an influential factor in occurrence of men infertility. AFB1 lower sensitivity of thyroid receptors by en- hanced generation of reactive oxygen spe- cies, aggravating lipid peroxidation concen- trations (15). Mycotoxins could be raised in animal and hu- man biological fluids after feeding of con- taminated food products. However, nowa- days contamination through inhalation of mycotoxins in indoor air has been taken in consideration (16). This study was proposed to clarify the possi- ble effects of aflatoxin B1 as an endocrine disruptor on pituitary gland, thyroid gland and gonads among miller flour workers, and to evaluate its effects on human male sexual function. Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 4 | 12 Methods Study design This was a case-control study. The exposed workers were considered as cases compared to the non-exposed subjects (controls). The variable fertility/sub-fertility in this study was measured through estimation of the sex hormones as high LH and FSH and low testosterone and rate of occurrence of sexual male function disorders among the ex- posed workers compared to their non-ex- posed can affect the fertility of the exposed workers. Study population and sampling This study included all the miller flour ex- posed male workers (42 workers) from the grinding department (which showed the high- est level of aspergillus flavus in the air sam- pling of airborne fungi). Forty male non ex- posed subjects were included in the study (they were all the available employees work- ing in the area surrounding the flour mill). Data collection Written informed consent was obtained from all the included subjects. Questionnaire was filled during personal interview with the par- ticipating groups. The questionnaire included (personal data, detailed occupational history, marital, sexual and medical history, types and use of personal protective equipment). Avail- able personal protective equipment included masks, protective goggles and gowns. Exclusion criteria were obesity, history of di- abetes, hypertension and thyroid diseases, which may be considered differential causes for infertility. Sampling of airborne fungi The samples were taken during the normal working days, between 9.00 am to 2.00 pm to determine peak exposures inside the flour mill. The air sampler was positioned at a height of ~ 1.5 m (breathing zone) above the floor level in the middle of the sampling lo- cation. The control(comparison) samples were taken 10 m outside the building. Andersen one-stage viable cascade impact or sampler (TE-10-160, Tisch Environmental Cleves, OH, USA) was used. It collects par- ticles with aerodynamic diameter of < 2.5 µm. Particles < 2.5 µm penetrate deeply into lungs. The sampler was operated at flow rate of 28.3 L/min for 5 min. Malt extract agar (MEA) were used to collect airborne fungi (BD BioSciences, Sparks, Maryland, USA). Three consecutive samples were taken during each sampling event (3 plates/location). Fungal plates were incubated at 28 °C for 5- 7 days and checked daily. The resultant colo- nies were counted and positive hole correc- tion was conducted on all counts prior to the calculation of the colony forming units per cubic meter of air (CFU/m3) (17). Fungal isolates were purified and identified by direct observation on the basis of micro and macro morphological features. Identifi- cation was performed on the basis of reverse and surface coloration of colonies on Sabouraud dextrose agar, CzapekDoxagar, potato dextrose agar and malt extract agar. Fungal isolates were identified to the genus or species level (18). Laboratory investigations -The blood samples were collected in sterile dry tubes, left to clot for 30 min and then cen- trifuged at 3000 r/min for 10 min. The sepa- rated sera were kept at -20 ˚C for the labora- tory investigations. -AFB1 and serum Alb:  Aflatoxin B1 was firstly extracted us- ing EASIEXTRACT1 aflatoxin im- mune affinity column (Scotland). AFB1 concentrations of the samples were analyzed by micro-titer plate en- zyme-linked immune-sorbent assay Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 5 | 12 (ELISA) method using RIDAS- CREEN1AFB1 30/15 ELISA, made in Germany.  Serum albumin (Alb) was determined by colorimetric method according to Doumas and Biggs (19). -Serum concentrations of LH,FSH, testos- terone, free triiodothyronine (fT3), free thy- roxin (fT4) and thyroid stimulating hormone (TSH) were measured using ELISA Kit by DRG International, Inc., (USA) at the re- search laboratory, 17-beta-estradiol (E2) us- ing kit by Biosource. Ethical approval number (10142) was ob- tained from the Research Ethics Committee of the National Research Centre, Egypt, be- fore the beginning of the study. Data analysis Statistical analysis was done through SPSS package version 20. Quantitative data were expressed as mean ± SD. Two independent sample T-test and Chi-square test were used to assess statistical differences in the quanti- tative and qualitative data (for distribution of sexual disorders among the studied groups) respectively between the exposed and the non-exposed groups. Pearson's correlation coefficient was calculated for exposure dura- tion, aflatoxin В1 and studied hormones among the exposed workers. P-values were two-tailed and considered statistically signif- icant at ≤0.05. Results Both studied groups were between 40 to 50 years, with mean age 45 ± 8.9 years for the exposed workers and 44± 9.2 years for the non-exposed group; without significant dif- ference. There was no significant difference between both studied groups regarding smok- ing habits; number of smokers was 26 among the exposed group and 24 among the non-ex- posed group. The mean of duration of expo- sure of the miller flour workers was 15 ± 5.2 years. None of the workers in the flour mill used personal protective equipments. Penicillium and Aspergillus were the com- mon airborne fungi in the flourmill. Penicil- lium and Aspergillus flavus concentrations ranged within39– 577 CFU/m3 and 12– 205 CFU/m3, respectively. Penicillium and As- pergillus flavus were found in the highest concentrations in the grinding unit. Aspergil- lus niger concentrations ranged from 19– 180 CFU/m3, with the highest concentration found in garbling unit. Table 1 shows Indoor/outdoor (I/O) ratio, "a relative standard" used to document the pres- ence or absence of indoor biologically de- rived contamination and differences between sampling sites (20). In the present study, I/O ratios for penicillium and aspergillus niger were ≤ 1 in almost all locations; suggesting that outdoor air was the main contaminant source. However, I/O ratios for aspergillus flavus were ≥ 1 in all the locations indicating presence of indoor sources Table 1. I/O ratios of the common airborne fungi at the flourmill units Locations Penicillium species Aspergillus fla- vus Aspergillus ni- ger Aspergillus spe- cies* Storage 0.07 6.68 0.5 - Garbling 0.2 6.67 0.75 0.42 Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 6 | 12 Grinding 1.12 17.83 0.12 0.42 Packaging 0.26 1.86 1.16 0.57 *Aspergillus species include: Aspergillus parasiticus, Aspergillus terreus and Aspergillus ochraceus. Table 2 shows that AFB1-Albumin (AFB1- Alb) level among the exposed group was sig- nificantly higher compared to the non-ex- posed. LH and FSH were significantly higher and testosterone was significantly lower among the exposed workers compared to the non-exposed. There was no significant differ- ence regarding E2 and the thyroid hormones between the two studied groups. Data in table 3 shows that among the exposed workers, AFB1/Alb is significantly positively corre- lated with LH and negatively correlated withfT4 & fT3. fT4 is significantly nega- tively correlated with the duration of expo- sure. TSH was significantly negatively corre- lated with LH and FSH, and positively corre- lated with testosterone. LH was positively correlated with FSH on one side and nega- tively correlated with testosterone on the other side. Twenty-five percent (10/40) of the non-exposed and 45.2% (19/42)of the ex- posed group complain of sexual disorders. Fifteen percent (6/40) of the non-exposed versus 7.1% (3/42) of the exposed workers complained of one sexual disorder. While 10% (4/40) of non-exposed versus 38.1% (16/42) of the exposed workers complained of more than one sexual disorder. Table 4 shows the distribution of various types of sexual disorders (decreased libido, impo- tence, premature ejaculation) which was higher in the exposed workers. Table 2. Comparison of AFB1-Albumin (AFB1-Alb) level, the male sex hormones, E2 and thyroid hormones between the two studied groups Non-exposed (=40) Exposed (=42) Independent t-test Mean SD Mean SD t-test P-value AFB1/ALB ng/g 0.04 0.01 0.06 0.02 4.658 P< 0.001 LH (3-12mIU/ml) 5.763 .1469 7.542 .3271 4.960 P< 0.001 FSH (2-10mIU/ml) 6.442 .2644 30.542 3.9841 6.036 P< 0.001 Testosterone (0.083 – 16ng/ml) 5.5268 .19092 4.0593 .47891 2.846 P= 0.006 17β-estradiol E2 (11.2-43.2pg/ml) 29.6789 1.96325 31.8016 2.70151 0.636 0.527 fT4 (0.93-1.7ng/dl) 1.33 0.17 1.32 0.22 1.52 0.880 fT3 (2-4.4pg/ml) 2.75 0.36 2.92 0.60 1.51 0.135 TSH (0.5-8.9uIU/ml) 1.89 0.55 2.11 0.67 1.59 0.117 Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 7 | 12 AFB1-Albumin (AFB1-Alb), LH= luteinizing hormone, FSH= follicle stimulating hormone, E2 =17β-estra- diol, fT4= free thyroxin, fT3= free triiodothyronine, TSH= thyroid stimulating hormone. Table 3. Relation between exposure duration, aflatoxin В1 and studied hormones among the exposed workers Exposure duration AFB1/ALB LH FSH Testosterone E2 Exposure du- ration r= - 0.2 -.121 -.053 .200 .060 LH r= -.121 0.3* - .779** -.322* -.067 FSH r= -.053 0.1 .779** - -.294 -.110 Testosterone r= .200 -0.1 -.322* -.294 - -.051 E2 r= .060 -0.03 -.067 -.110 -.051 - fT4 r= -.336* -0.3* .073 .097 -.020 .163 fT3 r= -.066 -0.3* .096 .004 -.050 .088 TSH r= -.063 -0.01 -.507** -.334* .342* -.161 ** p<0.01, * p<0.05, r= Pearson's correlation;AFB1-Albumin (AFB1-Alb), LH= luteinizing hormone, FSH= follicle stimulating hormone, E2 =17β-estradiol, fT4= free thyroxine, fT3= free triiodothyronine, TSH= thy- roid stimulating hormone. Table 4. Distribution of sexual disorders among the studied groups Sexual symptoms Non-exposed (40) Exposed (42) Chi-square Number % Number % P-value Decreased li- bido 3 7.5 10 23.8 0.04 * Impotence 5 12.5 12 28.6 0.07 Premature ejaculation) 7 17.5 17 40.5 0.02* Infertility 2 5 3 7.1 0.6 * P<0.05. Discussion In the present study, sampling of airborne fungi revealed that aspergillus flavus & pen- icillum were the predominant fungal types in the flour mill. Indoor/Outdoor ratios for as- pergillus flavus were ≥ 1 in all the locations indicating presence of indoor sources. Serum AFB1/Alb, LH and FSH, existence of various sexual disorders (decreased libido, impotence and premature ejaculation) were higher while testosterone was lower in the miller flour workers compared to non-exposed. However, there was no significant difference regarding 17-beta-estradiol E2 and thyroid hormone levels between both studied groups. Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 8 | 12 AFB1/Alb is significantly positively corre- lated with LH and negatively correlated with fT4 & fT3. fT4 is significantly negatively correlated with the duration of exposure. The results in the present study agree with Awad study (21) which found that Aspergil- lus and Penicillium were the dominant air- borne fungi in flourmill buildings. The concentrations of Aspergillus flavus in- side the different flour mill units exceeded outdoor ones. The dominance of Aspergillus flavus is an indication of inadequate storage conditions and high water content of grains (21). Contamination of different grains by Aspergillus niger, Aspergillus flavus and Fusariumoxysporium occurred due to poor environmental conditions during pre and postharvest of grains (22). In the present study, indoor/outdoor (I/O) ratios of Penicil- lium, Aspergillus niger and other Aspergillus species almost did not exceed 1, meaning that outdoor was the main source. However, I/O ratio of Aspergillus flavus exceeded 1, reached 17.83 (in grinding) indicating the presence of inside generative sources (23). AFB1, have great effect on the endocrine glands and reproductive system both in hu- mans and in experimental animals. However, literature on the effect of aflatoxin on human reproduction is scarce (24). Serum AFB1-Albadduct level was signifi- cantly higher among the miller flour workers compared to the non-exposed in the present study. AFB1-Alb level detection in serum is a reliable indicator of long-term exposure to aflatoxin (25). So, the rise of AFB1-Alb level among the workers could be attributed to their occupational exposure to relatively high concentrations of aspergillus flavus through inhalation by handling flour dust which rep- resents an additional exposure risk to those subjects than the general population, which was confirmed by the high I/O ratio of Asper- gillus flavus. The present study showed decreased serum testosterone and increased serum level of FSH and LH among the miller flour workers compared to the non-exposed. Moreover, there is positive correlation between LH &AFB1/ALB in the exposed group. These findings may be due to increase level of AFB1-Alb among the exposed workers than the non-exposed, which could be due to the high concentration of aspergillus flavus in the air environment of the flour mill in the pre- sent study. Previous studies (26,27) showed that concen- trations of serum FSH, LH and testosterone were reduced in AFB1 treated rats. Another study (28) found similar results in male chicken fed on different concentrations of di- etary aflatoxin. Results showed decreased se- rum testosterone levels and LH in the afla- toxin treated groups compared to the control group. Another author (29) administered AFB1orally in male rats for 48 days at differ- ent doses. The concentrations of serum LH and testosterone were lower, but on the other hand serum FSH was higher in the treated groups. After the administration of different doses of AFB1, the concentration of serum testos- terone was significantly reduced, in a dose- dependent manner in rabbit (30); in Japanese breed quails (31); in white leghorn male chicken (32) and in goats (33). The diversity of results of various experi- mental animal studies could be due to species variances or due to difference in route of ex- posure, potency or the dose of AFB1and the duration of exposure. In a previous study (34), the serum testos- terone concentration was significantly lower while the levels of serum FSH and LH in- creased significantly in adult rats exposed to AFB1 compared to non-exposed. These find- ings agreed with the results of the present study. Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 9 | 12 Verma and his colleague (35) supported that reduced serum testosterone concentration is attributed to mitochondria dysfunction, to in- hibition in protein synthesis or enzyme activ- ity or to membrane changes of leydig cells. They added that increased level of LH along with decreased level of serum testosterone in experimental rats exposed to AFB1 reflect decreased steroidogenic ability of the testes suggesting permanent changes in leydig cell function. The degenerative effect of the aflatoxin on germinal epithelium of the seminiferous tu- bules would breakout into sertoli cells, lead- ing to decrease in inhibin B1 level thus reduc- ing its inhibitory effect on secretion of FSH leading to its elevation (36). Direct effect of AFB1on leydig cells and ser- toli cells in the testes leading to reduction of the gonadal hormones; testosterone and estra- diol may be due to the action of AFB1 on binding of DNA to form complexes and inhi- bition of nucleic acid synthesis (32). In the current study, 45.2% of the exposed group versus 25% of the non-exposed group complained of sexual disorders. The distribu- tion of different types of sexual disorders (de- creased libido, impotence, premature ejacula- tion) was higher among the miller flour work- ers compared to non-exposed. This might be due to increase of (AFB1-Alb) level in the ex- posed group than in the non-exposed as some mycotoxins can act as probable endocrine disruptors and cause changes in hormone production (5) and can cause reproductive anomalies (morphological and functional gonadal dysfunction, e.g. infertility and de- creased libido) (7). Also, the decrease of tes- tosterone in the exposed group might be the cause of decreased libido, and potency in this group, as testosterone is necessary to main- tain male secondary sex characteristics, li- bido, and probably potency. Thus patients with endocrine abnormalities may present with variety of symptoms, elevated levels of the gonadotropins, FSH and LH in the pres- ence of decreased testosterone levels indicat- ing primary testicular dysfunction (37), which agreed with the results of the present study. In the present study, although there was no significant difference between the exposed and the non-exposed groups regarding the levels of the thyroid hormones, yet, there was negative correlation between fT4 and dura- tion of exposure, and between fT3, fT4 and AFB1-Alb. These findings might suggest thyroid gland affection by aflatoxin on the long run. Moreover, there was a negative correlation between TSH and both LH, FSH, and posi- tive correlation between TSH and testos- terone, which means that decrease of TSH level occurred with lowering of testosterone and elevating LH & FSH levels indicating in- tact hypothalamo-pituitary-thyroid axis. Limitation of the study Information on sexual health was assessed using self-reporting which is a source of in- formation bias. Further studies are needed to be done on a larger scale. Conclusion Our results showed that AFB1 causes altera- tions in the serum concentrations of the gon- adotropic (FSH and LH), as well as gonadal (testosterone) hormones in the form of signif- icant increase in the serum concentrations of LH and FSH, as well as significant decrease in testosterone levels among exposed work- ers. The lowered levels of testosterone with elevated levels of FSH and LH indicate intact pituitary testicular axis in AFB1 exposed workers. These findings may confirm the ability of AFB1as endocrine disruptor to af- fect human male reproductive health. That is why it is highly recommended to estimate the levels of both gonadotropic (FSH and LH) Beshir S, Shaheen W, Saad-Hussein A, Saeed Y. Aflatoxin B1 as an endocrine disruptor among miller flour workers (Original article). SEEJPH 2020, posted: 04 May 2020. DOI: 10.4119/seejph-3441 P a g e 10 | 12 hormones periodically in exposed workers to pick up any early changes in their levels. References 1. 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