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Salud Pública de México

versión impresa ISSN 0036-3634

Salud pública Méx vol.64 no.1 Cuernavaca ene./feb. 2022  Epub 06-Mar-2023 

Artículos originales

Aflatoxin levels and prevalence of TP53 aflatoxin-mutations in hepatocellular carcinomas in Mexico

Niveles de aflatoxina y prevalencia de mutaciones del gen TP53 por aflatoxina en carcinoma hepatocelular en México

Leonardo S Lino-Silva, MD1 

Martín Lajous, ScD2  3 

Marion Brochier, MD2 

Luis Santiago-Ruiz, MD2 

Javier Melchor-Ruan, MD4 

Yi Xie, PhD5 

Mingyi Wang, PhD6 

Dongjing Wu, PhD6 

Herbert Higson, PhD6 

Kristine Jones, PhD6 

Martin Romero-Martínez, PhD7 

Salvador Villalpando, PhD8 

Alejandro Mohar, ScD9 

Joshua W Smith, PhD10 

Christian S Alvarez, PhD5) 

Katherine A McGlynn, PhD5 

Michael Dean, PhD5 

John Groopman, PhD10 

(1) Departamento de Patología, Instituto Nacional de Cancerología. Mexico City, Mexico.

(2) Centro de Investigación en Salud Poblacional, Instituto Nacional de Salud Pública. Mexico City, Mexico.

(3) Department of Global Health and Population, Harvard T.H. Chan School of Public Health. Boston, Massachusetts, United States.

(4) Departamento de Cirugía Oncológica, Instituto Nacional de Cancerología. Mexico City, Mexico.

(5) Division of Cancer Epidemiology and Genetics, National Cancer Institute. Bethesda, Maryland, United States.

(6) Cancer Genetics Research Laboratory, Division of Cancer Epidemiology and Genetics, Leidos Biomedical Research Inc., Frederick National Laboratory for Cancer Research. Gaithersburg, Maryland, United States.

(7) Centro de Investigación en Evaluación y Encuestas, Instituto Nacional de Salud Pública. Cuernavaca, Morelos, Mexico.

(8) Centro de Investigación en Nutrición y Salud, Instituto Nacional de Salud Pública. Cuernavaca, Morelos, Mexico.

(9) Unidad de Investigación Biomédica en Cáncer (Instituto de Investigaciones Biomédicas, UNAM / Dirección de Investigación, Instituto Nacional de Cancerología). Mexico City, Mexico.

(10) Department of Environmental Health and Engineering, Bloomberg School of Public Health, Johns Hopkins University. Baltimore, United States.



To determine the exposure to aflatoxin B1 (AFB1) in southern Mexico and the presence of the aflatoxin signature mutation in hepatocellular carcinoma (HCC) tissue from patients from a cancer referral center.

Materials and methods:

We estimated the prevalence and distribution of AFB1 in a representative sample of 100 women and men from Chiapas using the National Health and Nutrition Survey 2018-19. We also examined the presence of the aflatoxin signature mutation in codon 249 (R249S), and other relevant mutations of the TP53 gene in HCC tissue blocks from 24 women and 26 men treated in a national cancer referral center.


The prevalence of AFB1 in serum samples was 85.5% (95%CI 72.1-93.1) and the median AFB1 was 0.117 pg/µL (IQR, 0.050-0.350). We detected TP53 R249S in three of the 50 HCCs (6.0%) and observed four other G>T transversions potentially induced by AFB1.


Our analysis provides evidence that AFB1 may have a relevant role on HCC etiology in Mexico.

Keywords: aflatoxin B1; mycotoxin; environmental biomarkers; mutation; liver neoplasia; epidemiology



Determinar la exposición a aflatoxina_B1 (AFB1) en el sur de México y la presencia de la mutación característica de AFB1 en tejido de carcinoma hepatocelular (CHC) de pacientes de un centro oncológico.

Material y métodos:

Se estimó la prevalencia y distribución de AFB1 en una muestra representativa de 100 mujeres y hombres de Chiapas a partir de la Encuesta Nacional de Salud y Nutrición 2018-19. También se observó la presencia de la mutación característica de AFB1 en el codón 249 (R249S), y otras mutaciones relevantes del gen TP53 en bloques de tejido de CHC de 24 mujeres y 26 hombres estudiados en un centro de referencia nacional de oncología.


La prevalencia de AFB1 en las muestras de suero fue de 85.5% (IC95% 72.1-93.1) y la mediana de la concentración 0.117 pg/µL (IQR, 0.050-0.350). Se detectó TP53 R249S en tres de 50 casos de CHC (6.0%) y se observaron cuatro transversiones G>T potencialmente inducidas por AFB1.


El presente análisis proporciona evidencia de que la AFB1 puede tener un papel relevante en la etiología del CHC en México.

Palabras clave: aflatoxina B1; micotoxinas; biomarcadores ambientales; mutación; neoplasias hepáticas; epidemiología


In Mexico, hepatocellular carcinoma (HCC), follows a unique epidemiologic pattern. Male-to-female mortality ratios are close to one.1 And Hepatitis B virus (HBV; 0.2%)2 and hepatitis C virus (HCV; 0.4%)3 seroprevalence are low. However, contamination of maize tortilla, a staple food in Mexico, with aflatoxin B1 (AFB1), a potent liver carcinogen, has been shown to be as high as 95% in store samples in Veracruz.4 And Chiapas and Guatemala, together represent the region with the highest HCC burden in the Americas. We aimed to estimate AFB1 exposure in a representative sample of adults from Chiapas.1 To further characterize the relevance of aflatoxin in Mexico, we determined the presence of the aflatoxin signature mutation in codon 249 (R249S) of the TP53 gene5 in HCC tissue from patients from a cancer referral center.

Materials and methods

National Health and Nutrition Survey, 2018-2019 (Ensanut 2018-19)

This nationally representative probabilistic multistage stratified cluster sampling survey (representative at the regional, state, urban/rural, and socioeconomic strata-level) obtained information from 44 069 households.6 A sample of 899 households from the state of Chiapas was probabilistically selected. Among 869 respondents (96.6%), we identified 308 individuals between 40 and 59 years of age (considered to be at highest risk for HCC)7 and randomly selected 100 serum samples from the 202 (65.5%) persons who donated a blood sample. Age, sex, education and rural residence distribution of selected participants was comparable to survey participants.

Measurement of AFB1 -lysine (AFB1 -lys)

We assessed AFB1 -lys (pg/uL serum) by taking 250μL of samples which were stored at -70°C. We then added 250μL of a Pronase-PBS (13mg mL-1) solution and incubated in water-bath at 37-40°C for 4.5h. Solid-phase extraction (SPE) was conducted on a Waters Oasis MAX 1cc 30mg extraction cartridge with a Phenomenex 24-port vacuum manifold. We conducted analyses using UPLC-TOF-MS/MS Waters, model Synapt G2 SI, equipped with a BEH C18 column (2.1x50μm, 1.7μm), with electrospray ionization in positive ion mode as previously described.8 Quantitation was performed using an 8-point, serially diluted, isotope dilution calibration curve in 25% aqueous methanol (v/v). The limits of detection and quantification were both 0.010 pg/μL.

Hepatocellular carcinoma tissue samples

We identified 61 women and 85 men from mostly Mexico City and surrounding areas treated in 2005-2015 for HCC at the Instituto Nacional de Cancerología for whom formalin-fixed paraffin-embedded HCC tissue blocks were available. We extracted date and place of birth, residence, HBV/HCV serostatus, regular alcohol use, ever smoking, diabetes diagnosis, and anthopometry from medical records. We confirmed the HCC diagnosis by central review. We included all women (n=26) and a random sample of 26 men (out of 40) with HCC risk factor information. We excluded two women born outside of Mexico.

DNA extraction and sequencing

Chilled blocks were sectioned until the entire tissue was accessed. Duplicate samples of two 10uM sections were collected, and the blade and microtome cleaned before the next sample was processed. FFPE (formalin-fixed, paraffin-embedded) sections were incubated with lysis buffer for 15 min at 80°C. Proteinase K was added, and samples incubated at 70°C overnight. The clear solution under the wax was removed and the DNA was extracted using Mag-Bind FFPE DNA kit (Omega Biotek, Norcross, GA) and eluted in 40 µl of ddH2O. DNA was quantitated using the Qubit Fluorometer dsDNA high sensitivity assay kit (ThermoFisher Scientific).

Targeted capture was performed on all exons of 245 known cancer-related genes using a NimbleGen capture array. Libraries were prepared with the Kapa HyperPlus kit, quantified using the PicoGreen dsDNA Reagent, normalized, and pooled. The pooled samples were captured with the custom NimbleGen Roche SeqCap EZ Choice custom panel, and 2x150bp sequencing was performed on either an Illumina HiSeq4000, or NovaSeq. Sequences were aligned, and TP53 mutations were identified. Variants passing quality control and filtering were visually confirmed using the Integrative Genomics Viewer (IGV). Variants observed 1 or 2 times, and likely to be enriched in somatic mutations were analyzed for mutational signatures using Mutagene.*

Statistical analysis

Median AFB1 -lys and interquartile range (IQR) were calculated prior to log10-transformation for other analyses. We estimated AFB1 exposure prevalence and geometric means and 95% confidence intervals (95%CI) using non-response-adjusted sampling weights based on probabilities of selection of households, individuals, and blood sample collection participants. We used linear regression to estimate the ratio of geometric means and 95%CIs across age, sex, habitual residence (urban/rural), ethnicity, and education and Poisson regression to estimate prevalence ratios (and 95%CIs) using Stata (StataCorp. 2015. Release 14. College Station, TX). The study was approved by the institutional review boards at Mexico’s National Institutes of Public Health and Cancer.


In a representative sample of adults aged 40-59 from Chiapas (mean age, 48.3 years; SD ± 6.0;) nearly 50% were from rural areas and approximately one third indigenous. The overall prevalence of detectable AFB1 -lys was 85.5% (95%CI 72.1, 93.1) representing 970 702 urban and rural individuals in Chiapas (table I). Prevalence appeared to be higher in younger, indigenous adults who live in rural areas. Median AFB1 was 0.117 pg/µL (IQR, 0.300) and the geometric mean 2.03 pg AFB1 -lys /µL (95%CI 1.11, 3.72; table II). Adduct levels were three-fold higher in men relative to women (3.68 vs.1.20 pg AFB1 -lys /µL) and in participants living in rural as compared to those in urban areas (3.67 vs.1.22 pg AFB1 -lys /µL).

Table I Prevalence and prevalence ratios of detected serum AFB1 -lys according to participant characteristics. Chiapas, Mexico, 2018-2019 




Prevalence ratio




(72.1, 93.1)





(75.5, 96.1)




(59.8, 91.7)


(0.73, 1.08)




(69.7, 94.7)




(66.2, 93.9)


(0.81, 1.16)




(75.6, 94.9)




(57.5, 94.7)


(0.74, 1.18)




(75.1, 95.3)




(46.2, 93.1)


(0.62, 1.21)


0-6 years


(66.9, 92.6)


≥7 years


(69.8, 97.0)


(0.89, 1.29)

Weighted n=1 135 324. Indigenous, participants who reported speaking an indigenous language. Sampling weights used for all estimates.

Table II Geometric means and geometric mean ratios of serum AFB1 -lys according to participant characteristics. Chiapas, Mexico, 2018-2019 



Geometric mean


Ratio of geometric mean




(1.11, 3.72)






(0.95, 7.86)





(1.06, 1.79)


(0.16, 1.51)





(1.00, 12.25)





(1.10, 1.42)


(0.10, 1.25)





(1.03, 14.57)





(1.09, 1.25)


(0.08, 1.13)





(1.04, 2.66)





(0.50, 23.05)


(0.27, 15.14)


0-6 years



(1.03, 6.23)


≥7 years



(1.13, 1.45)


(0.20, 1.25)

Weighted n=1 135 324. Indigenous, participants who reported speaking an indigenous language.

* In pg AFB1 -lys/µL. Sampling weights used for all estimates.

Characteristics of the 50 HCC patients are shown in table III. The median age was 63 years (interquartile range, 20). The prevalence of chronic HBV infection (HBsAg+) was 4.0% and the prevalence of HCV infection (anti-HCV+) was 14.0%. Overweight and obesity were common in both women and men. Cirrhosis was present in 25% of the women and 42.3% of the men. We detected the TP53 R249S mutation in three patients (all from mostly rural regions) of the 50 HCCs (6.0%). Two of them were from Veracruz and one was born in Oaxaca (but lived in Mexico City). Four patients with other TP53 G>T transversions (two samples with TP53 V157F, one with TP53 V203L, and one with TP53 G245V). In total, seven (14%) of patients had mutations that could be related to AFB1 exposure. Within this group, three tumors were from women and evidence of chronic HCV infection was present in only one patient. A preliminary analysis of mutational signatures revealed that up to 10% of somatic mutations in these tumors may be due to aflatoxin (figure 1).

Table III Characteristics of HCC patients included in the study (n=50) diagnosed between 2005-2015 in Mexico City by gender 


n= 24


n= 26

Median age, years (IQR)

64.5 (27)

63.0 (6)

Hepatitis B virus +

1 (4.2)

1 (3.8)

Hepatitis C virus +

3 (12.5)

4 (15.4)

Regular alcohol intake

3 (12.5)

17 (65.4)

Ever smoker

5 (20.8)

12 (46.1)

Mean BMI, kg/m² (± SD)

24.9 (5.4)

25.5 (3.3)

BMI categories

Normal weight, <25

13 (54.2)

15 (57.7)

Overweight, 25-29.9

7 (29.2)

7 (26.9)

Obese, ≥30

4 (16.6)

4 (15.4)


7 (29.2)

9 (34.6)

Missing information

6 (25.0)

3 (11.5)


6 (25.0)

11 (42.31)

Fatty liver

7 (29.2)

6 (23.1)



8 (33.4)

11 (42.3)


11 (45.8)

8 (30.8)


5 (20.8)

7 (26.9)


TP53 R249S

0 (0)

3 (11.5)

Other TP53 G>T*

3 (12.5)

1 (7.7)

IQR: interquartile range; BMI: body-mass index=weight in kilograms divided by height in meters squared; SD: standard deviation; HCC: hepatocellular carcinoma. Data presented as number (%) unless otherwise specified.

* includes TP53 V157F, V203L, and G245V.

* Mutations observed 1 or 2 times and likely enriched in somatic mutations were analyzed using Mutagene Identify (

Figure 1 Frequency of mutations* observed 1 or 2 times on 50 women and men with HCC diagnosed between 2005-2015 in Mexico City 


We found a high prevalence of exposure to aflatoxin in urban and rural Chiapas. However, circulating AFB1 levels were moderate. Also, aflatoxin-associated TP53 R249S mutation prevalence was moderate relative to high aflatoxin exposure regions in the world.

In Chiapas, AFB1 exposure was >85%. While the magnitude of the exposure was important (0.117 pg/µL; 2.8 pg/mg), it was not as high as in Guatemala (in the Southern border of Chiapas) where AFB1 was detectable in all participants and the median AFB1 -lys was 8.4 pg/mg (23.8 conversion factor from pg/uL to pg/mg).9 An initial AFB1 estimate in adult indigenous women in Mexico found widespread exposure but at a more moderate level relative to very high HCC burden areas.10 While the study included women mostly in their early thirties, our results may be partly consistent with that observation. However, the exposure levels reported in our study in adults currently living in Chiapas confer increased risk of HCC.11

AFB1 undergoes enzymatic conversion in the liver by CYP3A4 to the AFB1 -8,9-epoxide, the active metabolite that reacts with DNA to form an AFB-N7-guanine adduct. These adducts principally cause G:T/C:A mutations on the transcribed strand through the transcription-coupled pathway of nucleotide excision repair.12 Almost two decades ago a small study in Northern Mexico documented the presence of the AFB1 codon 249 mutations in HCC tissue (19% prevalence).13 In another study, this mutation was not present in any of the 69 HCC tissue samples from highly selected patient population in Mexico City most of whom with cirrhosis.14 The prevalence of this mutation in our study was lower that what was observed then and in Guatemala (24% prevalence) but higher than the 1-3% seen in low aflatoxin regions (the US, Europe, Korea).5,15 While R249S is the dominant TP53 aflatoxin-induced mutation, this mutation has been studied almost exclusively in high HBV prevalence regions. We also saw several examples of mutation G T/C:A in V157F. This mutation is another hotspot in TP53, but has not been causally linked to aflatoxin exposure in addition, R249S and V157F have been reported to be associated with a higher stem-cell-like gene expression and poorer survival.16

Major strengths of our analyses are generalizability of AFB1 exposure estimates to adults in Chiapas and detailed sequencing of TP53 mutations. However, AFB1 -lys analyses were done in a limited number of participants affecting confidence on subgroup analyses. The examination of the TP53 mutations was performed in HCC cases that may not be generalizable to all patients seen in Mexico. Currently most HCC cases are diagnosed using imaging without pathology confirmation and, similar to other countries, only a fraction of HCCs were biopsied. Also, the patient population included in our study may not adequately represent the source population of HCC patients in Mexico. In conclusion as the HCC burden remains understudied, our analysis provides evidence that in Mexico AFB1 may have a potentially important role in the burden of HCC. The five areas in Mexico with the highest HCC burden in order are Chiapas, Veracruz, Yucatán, Tabasco, and Campeche. Future research should focus in these geographic areas and accurately estimate aflatoxin exposure, identify subpopulations at risk and characterize sources of exposures.


This research was funded by the US National Cancer Institute’s Intramural Research program and grant P20CA210286. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute.


Global Burden of Disease Collaborative Network. GBD Results Tool [Internet]. GHdx, 2019 [cited October 26, 2021]. Available from:Available from: ]

Schweitzer A, Horn J, Mikolajczyk RT, Krause G, Ott JJ. Estimations of worldwide prevalence of chronic hepatitis B virus infection: a systematic review of data published between 1965 and 2013. Lancet. 2015;386(10003):1546-55. [ Links ]

Blach S, Collaborators POH. Global prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study. Lancet Gastroenterol Hepatol. 2017;2(3):161-76. [ Links ]

Wall-Martinez HA, Ramirez-Martinez A, Wesolek N, Brabet C, Durand N, Rodriguez-Jimenes GC, et al. Risk assessment of exposure to mycotoxins (aflatoxins and fumonisins) through corn tortilla intake in Veracruz City (Mexico). Food additives & Contaminants. 2019;36(6):929-39. [ Links ]

International Agency for Research on Cancer. TP53 Mutation Database [Internet]. Lyon: International Agency for Research on Cancer, 2017. Available from:http://www-p53.iarc.frLinks ]

Romero-Martínez M, Shamah-Levy T, Vielma-Orozco E, Heredia-Hernández O, Mojica-Cuevas J, Cuevas-Nasu L, et al. National Health and Nutrition Survey 2018-19: methodology and perspectives. Salud Publica Mex. 2019;61(6):917-23. [ Links ]

Groopman JD. Environmental health in the biology century: Transitions from population to personalized prevention. Experimental Biology and Medicine. 2019;244(9):728-33. [ Links ]

McCoy LF, Scholl PF, Schleicher RL, Groopman JD, Powers CD, Pfeiffer CM. Analysis of aflatoxin B1-lysine adduct in serum using isotope-dilution liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom. 2005;19(16):2203-10. [ Links ]

Smith JW, Kroker-Lobos MF, Lazo M, Rivera-Andrade A, Egner PA, Wedemeyer H, et al. Aflatoxin and viral hepatitis exposures in Guatemala: Molecular biomarkers reveal a unique profile of risk factors in a region of high liver cancer incidence. PLoS One. 2017;12(12):e0189255. [ Links ]

Diaz de Leon-Martinez L, Diaz-Barriga F, Barbier O, Ortiz DLG, Ortega-Romero M, Perez-Vazquez F, et al. Evaluation of emerging biomarkers of renal damage and exposure to aflatoxin-B1 in Mexican indigenous women: a pilot study. Environ Sci Pollut Res Int. 2019;26(12):12205-16. [ Links ]

Chen JG, Egner PA, Ng D, Jacobson LP, Muñoz A, Zhu YR, et al. Reduced aflatoxin exposure presages decline in liver cancer mortality in an endemic region of China. Cancer Prevention Research. 2013;6(10):1038-45. [ Links ]

Bedard LL, Massey TE. Aflatoxin B1-induced DNA damage and its repair. Cancer Lett. 2006;241(2):174-83. [ Links ]

Soini Y, Chia SC, Bennett WP, Groopman JD, Wang JS, DeBenedetti VM, et al. An aflatoxin-associated mutational hotspot at codon 249 in the p53 tumor suppressor gene occurs in hepatocellular carcinomas from Mexico. Carcinogenesis. 1996;17(5):1007-12. [ Links ]

Valdés-Peregrina EN, Sánchez-Hernández BE, Gamboa-Domínguez A. Metabolic syndrome instead of aflatoxin-related TP53 R249S mutation as a hepatocellular carcinoma risk factor. Rev Invest Clin. 2020;72(5):316-22. [ Links ]

Alvarez CS, Ortiz J, Bendfeldt-Avila G, Xie Y, Wang M, Wu D, et al. Analysis of TP53 aflatoxin signature mutation in hepatocellular carcinomas from Guatemala: A cross-sectional study (2016-2017). Health Science Reports. 2020;3(2):e155. [ Links ]

Woo HG, Wang XW, Budhu A, Kim YH, Kwon SM, Tang ZY, et al. Association of TP53 mutations with stem cell-like gene expression and survival of patients with hepatocellular carcinoma. Gastroenterology. 2011;140(3):1063-70. [ Links ]

Received: August 26, 2021; Accepted: November 25, 2021; Published: February 25, 2022

Corresponding author: Martín Lajous. Instituto Nacional de Salud Pública. 7ª Cerrada Fray Pedro de Gante 50.14000 CDMX, Mexico email:

Declaration of conflict of interests. The authors declare that they have no conflict of interests.

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