Hostname: page-component-cd9895bd7-8ctnn Total loading time: 0 Render date: 2024-12-23T19:18:10.320Z Has data issue: false hasContentIssue false
  • English
  • Français

Prolonged atrazine exposure beginning in utero and adult uterine morphology in mice

Published online by Cambridge University Press:  30 March 2021

Meaghan J. Griffiths Open the ORCID record for Meaghan J. Griffiths [Opens in a new window] ,
Amy L. Winship Open the ORCID record for Amy L. Winship [Opens in a new window] ,
Jessica M. Stringer Open the ORCID record for Jessica M. Stringer [Opens in a new window] ,
Elyse O. Swindells ,
Alesia P. Harper ,
Bethany J. Finger ,
Karla J. Hutt Open the ORCID record for Karla J. Hutt [Opens in a new window]  and
Mark P. Green Open the ORCID record for Mark P. Green [Opens in a new window]
Meaghan J. Griffiths
Affiliation:
Development and Stem Cells Program, Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
Amy L. Winship
Affiliation:
Development and Stem Cells Program, Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
Jessica M. Stringer
Affiliation:
Development and Stem Cells Program, Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
Elyse O. Swindells
Affiliation:
Development and Stem Cells Program, Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
Alesia P. Harper
Affiliation:
School of BioSciences, University of Melbourne, Parkville, Australia
Bethany J. Finger
Affiliation:
School of BioSciences, University of Melbourne, Parkville, Australia
Karla J. Hutt
Affiliation:
Development and Stem Cells Program, Department of Anatomy and Developmental Biology, Biomedicine Discovery Institute, Monash University, Clayton, Australia
Mark P. Green*
Affiliation:
School of BioSciences, University of Melbourne, Parkville, Australia
*
Address for correspondence: Dr Mark Green, School of BioSciences, University of Melbourne, Parkville, Melbourne, VIC 3010, Australia. Email: [email protected]
Get access Rights & Permissions [Opens in a new window]

Abstract

Through drinking water, humans are commonly exposed to atrazine, a herbicide that acts as an endocrine and metabolic disruptor. It interferes with steroidogenesis, including promoting oestrogen production and altering cell metabolism. However, its precise impact on uterine development remains unknown. This study aimed to determine the effect of prolonged atrazine exposure on the uterus. Pregnant mice (n = 5/group) received 5 mg/kg body weight/day atrazine or DMSO in drinking water from gestational day 9.5 until weaning. Offspring continued to be exposed until 3 or 6 months of age (n = 5–9/group), when uteri were collected for morphological and molecular analyses and steroid quantification. Endometrial hyperplasia and leiomyoma were evident in the uteri of atrazine-exposed mice. Uterine oestrogen concentration, oestrogen receptor expression, and localisation were similar between groups, at both ages (P > 0.1). The expression and localisation of key epithelial-to-mesenchymal transition (EMT) genes and proteins, critical for tumourigenesis, remained unchanged between treatments, at both ages (P > 0.1). Hence, oestrogen-mediated changes to established EMT markers do not appear to underlie abnormal uterine morphology evident in atrazine exposure mice. This is the first report of abnormal uterine morphology following prolonged atrazine exposure starting in utero, it is likely that the abnormalities identified would negatively affect female fertility, although mechanisms remain unknown and require further study.

Keywords

Endocrine-disrupting chemicalatrazineendometriumuterusoestrogen
Type
Original Article
Information
Journal of Developmental Origins of Health and Disease , Volume 13 , Issue 1 , February 2022 , pp. 39 - 48
Check for updates
Copyright
© The Author(s), 2021. Published by Cambridge University Press in association with International Society for Developmental Origins of Health and Disease

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Gysin, H, Knusli, E. Chemistry and herbicidal properties of triazine derivatives. Adv in Pest Control Res. 1960; 3, 289–353.Google Scholar
Radcliffe, JC. Pesticide Use in Australia: A Review Undertaken by the Australian Academy of Technological Sciences and Engineering, 2002. Australian Academy of Technological Sciences and Engineering, Melbourne, Victoria, Australia.Google Scholar
Cheremisinoff, NP, Rosenfeld, PE. Handbook of Pollution Prevention and Cleaner Production: Best Practices in the Agrochemical Industry, 2010; pp. 215–231. Elsevier Inc., Oxford, UK.Google Scholar
Allinson, G, Zhang, P, Bui, A, Allinson, M, Rose, G, Marshall, S, et al. Pesticide and trace metal occurrence and aquatic benchmark exceedances in surface waters and sediments of urban wetlands and retention ponds in Melbourne, Australia. Environ Sci Pollut Res Int. 2015; 22(13), 10214–10226.CrossRefGoogle ScholarPubMed
WHO. World Health Organization Report: Atrazine and Its Metabolites in Drinking Water, 2010. https://www.who.int/water_sanitation_health/publications/antrazine/en/.Google Scholar
Lewis, SE, Brodie, JE, Bainbridge, ZT, Rohde, KW, Davis, AM, Masters, BL, et al. Herbicides: a new threat to the Great Barrier Reef. Environ Pollut. 2009; 157(8–9), 2470–2484.CrossRefGoogle ScholarPubMed
Magnusson, M, Heimann, K, Ridd, M, Negri, AP. Pesticide contamination and phytotoxicity of sediment interstitial water to tropical benthic microalgae. Water Res. 2013; 47(14), 5211–5221.CrossRefGoogle ScholarPubMed
Vogel, J, Linard, J. Agricultural herbicide transport in a first-order intermittent stream, Nebraska, USA. Appl Eng Agric 2011; 27(1), 63–74.CrossRefGoogle Scholar
Wagner, U. Detection of phosphate ester pesticides and the triazine herbicide atrazine in human milk, cervical mucus, follicular and sperm fluid. Fresenius J Anal Chem. 1990; 337, 77–78.Google Scholar
Chevrier, C, Limon, G, Monfort, C, Rouget, F, Garlantezec, R, Petit, C, et al. Urinary biomarkers of prenatal atrazine exposure and adverse birth outcomes in the PELAGIE birth cohort. Environ Health Perspect 2011; 119(7), 1034–1041.CrossRefGoogle ScholarPubMed
Ochoa-Acuna, H, Frankenberger, J, Hahn, L, Carbajo, C. Drinking-water herbicide exposure in Indiana and prevalence of small-for-gestational-age and preterm delivery. Environ Health Perspect. 2009; 117(10), 1619–10624.CrossRefGoogle ScholarPubMed
Patisaul, HB. Effects of environmental endocrine disruptors and phytoestrogens on the kisspeptin system. Adv Exp Med Biol. 2013; 784, 455–479.CrossRefGoogle ScholarPubMed
Costa, EM, Spritzer, PM, Hohl, A, Bachega, TA. Effects of endocrine disruptors in the development of the female reproductive tract. Arq Bras Endocrinol Metabol. 2014; 58(2), 153–161.CrossRefGoogle ScholarPubMed
Cragin, LA, Kesner, JS, Bachand, AM, Barr, DB, Meadows, JW, Krieg, EF, et al. Menstrual cycle characteristics and reproductive hormone levels in women exposed to atrazine in drinking water. Environ Res. 2011; 111(8), 1293–1301.CrossRefGoogle ScholarPubMed
Farr, SL, Cooper, GS, Cai, J, Savitz, DA, Sandler, DP. Pesticide use and menstrual cycle characteristics among premenopausal women in the Agricultural Health Study. Am J Epidemiol. 2004; 160(12), 1194–11204.CrossRefGoogle ScholarPubMed
Freeman, LE, Rusiecki, JA, Hoppin, JA, Lubin, JH, Koutros, S, Andreotti, G, et al. Atrazine and cancer incidence among pesticide applicators in the agricultural health study (1994–2007). Environ Health Perspect. 2011; 119(9), 1253–1259.CrossRefGoogle Scholar
Boffetta, P, Adami, HO, Berry, SC, Mandel, JS. Atrazine and cancer: a review of the epidemiologic evidence. Eur J Cancer Prev. 2013; 22(2), 169–180.CrossRefGoogle ScholarPubMed
Jowa, L, Howd, R. Should atrazine and related chlorotriazines be considered carcinogenic for human health risk assessment? J Environ Sci Health C Environ Carcinog Ecotoxicol Rev. 2011; 29(2), 91–144.CrossRefGoogle ScholarPubMed
Cooper, RL, Laws, SC, Das, PC, Narotsky, MG, Goldman, JM, Lee Tyrey, E, et al. Atrazine and reproductive function: mode and mechanism of action studies. Birth Defects Res B Dev Reprod Toxicol. 2007; 80(2), 98–112.CrossRefGoogle ScholarPubMed
Jin, Y, Wang, L, Fu, Z. Oral exposure to atrazine modulates hormone synthesis and the transcription of steroidogenic genes in male peripubertal mice. Gen Comp Endocrinol. 2013; 184, 120–127.CrossRefGoogle ScholarPubMed
Mnif, W, Hassine, AI, Bouaziz, A, Bartegi, A, Thomas, O, Roig, B. Effect of endocrine disruptor pesticides: a review. Int J Environ Res Public Health. 2011; 8(6), 2265–2303.CrossRefGoogle ScholarPubMed
Kabir, ER, Rahman, MS, Rahman, I. A review on endocrine disruptors and their possible impacts on human health. Environ Toxicol Pharmacol. 2015; 40(1), 241–258.CrossRefGoogle ScholarPubMed
Roberge, M, Hakk, H, Larsen, G. Atrazine is a competitive inhibitor of phosphodiesterase but does not affect the estrogen receptor. Toxicol Lett. 2004; 154(1–2), 61–68.CrossRefGoogle Scholar
Holloway, AC, Anger, DA, Crankshaw, DJ, Wu, M, Foster, WG. Atrazine-induced changes in aromatase activity in estrogen sensitive target tissues. J Appl Toxicol. 2008; 28(3), 260–270.CrossRefGoogle ScholarPubMed
Kniewald, J, Osredecki, V, Gojmerac, T, Zechner, V, Kniewald, Z. Effect of s-triazine compounds on testosterone metabolism in the rat prostate. J Appl Toxicol. 1995; 15(3), 215–218.CrossRefGoogle ScholarPubMed
Fan, W, Yanase, T, Morinaga, H, Gondo, S, Okabe, T, Nomura, M, et al. Atrazine-induced aromatase expression is SF-1 dependent: implications for endocrine disruption in wildlife and reproductive cancers in humans. Environ. Health Perspect. 2007; 115(5), 720–727.CrossRefGoogle ScholarPubMed
Azam, S, Lange, T, Huynh, S, Aro, AR, von Euler-Chelpin, M, Vejborg, I, et al. Hormone replacement therapy, mammographic density, and breast cancer risk: a cohort study. Cancer Causes Control. 2018; 29(6), 495–505.CrossRefGoogle ScholarPubMed
Kotsopoulos, J, Gronwald, J, Karlan, BY, Huzarski, T, Tung, N, Moller, P, et al. Hormone replacement therapy after oophorectomy and breast cancer risk among BRCA1 mutation carriers. JAMA Oncol. 2018; 4(8), 1059–1065.CrossRefGoogle ScholarPubMed
Wang, K, Li, F, Chen, L, Lai, YM, Zhang, X, Li, HY. Change in risk of breast cancer after receiving hormone replacement therapy by considering effect-modifiers: a systematic review and dose-response meta-analysis of prospective studies. Oncotarget. 2017; 8(46), 81109–81124.CrossRefGoogle ScholarPubMed
Cooper, RL, Stoker, TE, Tyrey, L, Goldman, JM, McElroy, WK. Atrazine disrupts the hypothalamic control of pituitary-ovarian function. Toxicol Sci. 2000; 53(2), 297–307.CrossRefGoogle ScholarPubMed
Foradori, CD, Sawhney Coder, P, Tisdel, M, Yi, KD, Simpkins, JW, Handa, RJ, et al. The effect of atrazine administered by gavage or in diet on the LH surge and reproductive performance in intact female Sprague-Dawley and Long Evans rats. Birth Defects Res B Dev Reprod Toxicol. 2014; 101(3), 262–275.CrossRefGoogle ScholarPubMed
Bray, F, Ferlay, J, Soerjomataram, I, Siegel, RL, Torre, LA, Jemal, A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018; 68(6), 394–424.CrossRefGoogle ScholarPubMed
Soliman, PT, Oh, JC, Schmeler, KM, Sun, CC, Slomovitz, BM, Gershenson, DM, et al. Risk factors for young premenopausal women with endometrial cancer. Obstet Gynecol. 2005; 105(3), 575–580.CrossRefGoogle ScholarPubMed
Di Cristofano, A, Ellenson, LH. Endometrial carcinoma. Annu Rev Pathol. 2007; 2, 57–85.CrossRefGoogle ScholarPubMed
Murali, R, Soslow, RA, Weigelt, B. Classification of endometrial carcinoma: more than two types. Lancet Oncol. 2014; 15(7), e268–e278.CrossRefGoogle ScholarPubMed
NRMMC Na. Australian Drinking Water Guidelines, 2011. Canberra, ACT, Australia: Australian Government, pp. 1–1163.Google Scholar
Cook, LE, Finger, BJ, Green, MP, Pask, AJ. Exposure to atrazine during puberty reduces sperm viability, increases weight gain and alters the expression of key metabolic genes in the liver of male mice. Reprod Fertil Dev. 2019; 31(5), 920–931.CrossRefGoogle ScholarPubMed
Lin, Z, Fisher, JW, Wang, R, Ross, MK, Filipov, NM. Estimation of placental and lactational transfer and tissue distribution of atrazine and its main metabolites in rodent dams, fetuses, and neonates with physiologically based pharmacokinetic modeling. Toxicol Appl Pharmacol. 2013; 273(1), 140–158.CrossRefGoogle ScholarPubMed
Newbold, RR, Jefferson, WN, Padilla-Banks, E. Long-term adverse effects of neonatal exposure to bisphenol A on the murine female reproductive tract. Reprod Toxicol. 2007; 24(2), 253–258.CrossRefGoogle ScholarPubMed
Winship, AL, Gazzard, SE, Cullen-McEwen, LA, Bertram, JF, Hutt, KJ. Maternal low-protein diet programmes low ovarian reserve in offspring. Reproduction (Cambridge, England). 2018; 156(4), 299–311.Google ScholarPubMed
Winship, AL, Carpenter, M, Griffiths, M, Hutt, KJ. Vincristine chemotherapy induces atresia of growing ovarian follicles in mice. Toxicol Sci: Off J Soc Toxicol. 2019; 169(1), 43–53.CrossRefGoogle ScholarPubMed
Griffiths, M, Van Sinderen, M, Rainczuk, K, Dimitriadis, E. miR-29c overexpression and COL4A1 downregulation in infertile human endometrium reduces endometrial epithelial cell adhesive capacity in vitro implying roles in receptivity. Sci Rep. 2019; 9(1), 8644.CrossRefGoogle ScholarPubMed
Van Sinderen, M, Oyanedel, J, Menkhorst, E, Cuman, C, Rainczuk, K, Winship, A, et al. Soluble delta-like ligand 1 alters human endometrial epithelial cell adhesive capacity. Reprod Fertil Dev. 2015; 29(4), 694–702.CrossRefGoogle Scholar
Bilyk, O, Coatham, M, Jewer, M, Postovit, LM. Epithelial-to-mesenchymal transition in the female reproductive tract: from normal functioning to disease pathology. Front Oncol. 2017; 7, 145.CrossRefGoogle ScholarPubMed
Pfaffl, MW. Relative quantification. In Real-time PCR (ed. Tevfik Dorak M), 2007; pp. 89–108. Taylor & Francis, New York, USA.Google Scholar
McNamara, KM, Harwood, DT, Simanainen, U, Walters, KA, Jimenez, M, Handelsman, DJ. Measurement of sex steroids in murine blood and reproductive tissues by liquid chromatography-tandem mass spectrometry. J Steroid Biochem Mol Biol. 2010; 121(3–5), 611–618.CrossRefGoogle ScholarPubMed
Kniewald, J, Jakominic, M, Tomljenovic, A, Simic, B, Romac, P, Vranesic, D, et al. Disorders of male rat reproductive tract under the influence of atrazine. J Appl Toxicol. 2000; 20(1), 61–68.3.0.CO;2-3>CrossRefGoogle ScholarPubMed
Podsypanina, K, Ellenson, LH, Nemes, A, Gu, J, Tamura, M, Yamada, KM, et al. Mutation of Pten/Mmac1 in mice causes neoplasia in multiple organ systems. Proc Natl Acad Sci U S A. 1999; 96(4), 1563–1568.CrossRefGoogle ScholarPubMed
Ashby, J, Tinwell, H, Stevens, J, Pastoor, T, Breckenridge, CB. The effects of atrazine on the sexual maturation of female rats. Regul Toxicol Pharmacol. 2002; 35(3), 468–473.CrossRefGoogle ScholarPubMed
Laws, SC, Ferrell, JM, Stoker, TE, Schmid, J, Cooper, RL. The effects of atrazine on female wistar rats: an evaluation of the protocol for assessing pubertal development and thyroid function. Toxicol Sci. 2000; 58(2), 366–376.CrossRefGoogle ScholarPubMed
Tennant, MK, Hill, DS, Eldridge, JC, Wetzel, LT, Breckenridge, CB, Stevens, JT. Possible antiestrogenic properties of chloro-s-triazines in rat uterus. J Toxicol Environ Health. 1994; 43(2), 183–196.CrossRefGoogle ScholarPubMed
Quignot, N, Arnaud, M, Robidel, F, Lecomte, A, Tournier, M, Cren-Olivé, C, et al. Characterization of endocrine-disrupting chemicals based on hormonal balance disruption in male and female adult rats. Reprod Toxicol. 2012; 33(3), 339–352.CrossRefGoogle ScholarPubMed
Yang, CH, Almomen, A, Wee, YS, Jarboe, EA, Peterson, CM, Janát-Amsbury, MM. An estrogen-induced endometrial hyperplasia mouse model recapitulating human disease progression and genetic aberrations. Cancer Med. 2015; 4(7), 1039–1050.CrossRefGoogle ScholarPubMed
Hu, K, Tian, Y, Du, Y, Huang, L, Chen, J, Li, N, et al. Atrazine promotes RM1 prostate cancer cell proliferation by activating STAT3 signaling. Int J Oncol. 2016; 48(5), 2166–2174.CrossRefGoogle ScholarPubMed
Powell, ER, Faldladdin, N, Rand, AD, Pelzer, D, Schrunk, EM, Dhanwada, KR. Atrazine exposure leads to altered growth of HepG2 cells. Toxicol In Vitro. 2011; 25(3), 644–651.CrossRefGoogle ScholarPubMed
Sagarkar, S, Gandhi, D, Devi, SS, Sakharkar, A, Kapley, A. Atrazine exposure causes mitochondrial toxicity in liver and muscle cell lines. Indian J Pharmacol. 2016; 48(2), 200–207.Google ScholarPubMed
Bai, X, Sun, C, Xie, J, Song, H, Zhu, Q, Su, Y, et al. Effects of atrazine on photosynthesis and defense response and the underlying mechanisms in Phaeodactylum tricornutum. Environ Sci Pollut Res Int. 2015; 22(22), 17499–17507.CrossRefGoogle ScholarPubMed
Mendoza-Rodriguez, CA, Garcia-Guzman, M, Baranda-Avila, N, Morimoto, S, Perrot-Applanat, M, Cerbon, M. Administration of bisphenol A to dams during perinatal period modifies molecular and morphological reproductive parameters of the offspring. Reprod Toxicol. 2011; 31(2), 177–183.CrossRefGoogle ScholarPubMed
Rivera, OE, Varayoud, J, Rodriguez, HA, Munoz-de-Toro, M, Luque, EH. Neonatal exposure to bisphenol A or diethylstilbestrol alters the ovarian follicular dynamics in the lamb. Reprod Toxicol. 2011; 32(3), 304–312.CrossRefGoogle ScholarPubMed
Iguchi, T, Fukazawa, Y, Uesugi, Y, Takasugi, N. Polyovular follicles in mouse ovaries exposed neonatally to diethylstilbestrol in vivo and in vitro. Biol Reprod. 1990; 43(3), 478–484.CrossRefGoogle ScholarPubMed
Susiarjo, M, Hassold, TJ, Freeman, E, Hunt, PA. Bisphenol A exposure in utero disrupts early oogenesis in the mouse. PLoS Genet. 2007; 3(1), e5.CrossRefGoogle ScholarPubMed
Rodriguez, HA, Santambrosio, N, Santamaria, CG, Munoz-de-Toro, M, Luque, EH. Neonatal exposure to bisphenol A reduces the pool of primordial follicles in the rat ovary. Reprod Toxicol. 2010; 30(4), 550–557.CrossRefGoogle ScholarPubMed
Cook, J, Davis, B, Goewey, J, Berry, T, Walker, C. Identification of a sensitive period for developmental programming that increases risk for uterine leiomyoma in Eker rats. Reprod Sci. 2007; 14(2), 121–136.CrossRefGoogle ScholarPubMed
Prusinski Fernung, LE, Yang, Q, Sakamuro, D, Kumari, A, Mas, A, Al-Hendy, A. Endocrine disruptor exposure during development increases incidence of uterine fibroids by altering DNA repair in myometrial stem cells. Biol Reprod. 2018; 99(4), 735–748.Google ScholarPubMed
Mahalingaiah, S, Hart, JE, Wise, LA, Terry, KL, Boynton-Jarrett, R, Missmer, SA. Prenatal diethylstilbestrol exposure and risk of uterine leiomyomata in the Nurses’ Health Study II. Am J Epidemiol. 2014; 179(2), 186–191.CrossRefGoogle ScholarPubMed
Newbold, RR, Bullock, BC, McLachlan, JA. Uterine adenocarcinoma in mice following developmental treatment with estrogens: a model for hormonal carcinogenesis. Cancer Res. 1990; 50(23), 7677–7681.Google Scholar
Newbold, RR, Banks, EP, Bullock, B, Jefferson, WN. Uterine adenocarcinoma in mice treated neonatally with genistein. Cancer Res. 2001; 61(11), 4325–4328.Google ScholarPubMed
Suen, AA, Jefferson, WN, Wood, CE, Padilla-Banks, E, Bae-Jump, VL, Williams, CJ. SIX1 oncoprotein as a biomarker in a model of hormonal carcinogenesis and in human endometrial cancer. Mol Cancer Res. 2016; 14(9), 849–858.CrossRefGoogle Scholar
Branham, WS, Sheehan, DM. Ovarian and adrenal contributions to postnatal growth and differentiation of the rat uterus. Biol Reprod. 1995; 53(4), 863–872.CrossRefGoogle ScholarPubMed
Cupp, AS, Uzumcu, M, Suzuki, H, Dirks, K, Phillips, B, Skinner, MK. Effect of transient embryonic in vivo exposure to the endocrine disruptor methoxychlor on embryonic and postnatal testis development. J Androl. 2003; 24(5), 736–745.CrossRefGoogle Scholar
Anway, MD, Memon, MA, Uzumcu, M, Skinner, MK. Transgenerational effect of the endocrine disruptor vinclozolin on male spermatogenesis. J Androl. 2006; 27(6), 868–879.CrossRefGoogle ScholarPubMed
McBirney, M, King, SE, Pappalardo, M, Houser, E, Unkefer, M, Nilsson, E, et al. Atrazine induced epigenetic transgenerational inheritance of disease, lean phenotype and sperm epimutation pathology biomarkers. PLoS One. 2017; 12(9), e0184306.CrossRefGoogle ScholarPubMed
Greathouse, KL, Bredfeldt, T, Everitt, JI, Lin, K, Berry, T, Kannan, K, et al. Environmental estrogens differentially engage the histone methyltransferase EZH2 to increase risk of uterine tumorigenesis. Mol Cancer Res. 2012; 10(4), 546–557.CrossRefGoogle ScholarPubMed
NHMRC. Australian Code for the Care and Use of Animals for Scientific Purposes, 8th edition, 2013; pp. 1–96. National Health and Medical Research Council (NHMRC), Canberra ACT, Australia.Google Scholar
Supplementary material: Image

Griffiths et al. supplementary material

Griffiths et al. supplementary material

Download Griffiths et al. supplementary material(Image)
Image 7.7 KB