The interactions and combined effects of benzo[a]pyrene with other environmental pollutants

Summary

Benzo [a] pyrene (BaP) is a ubiquitous environmental carcinogen that poses growing health risks when co-exposed with other pollutants. However, there is still a poor understanding of the underlying interaction mechanisms, which hinders accurate risk assessment and targeted prevention. Co-exposure of BaP with heavy metals, particulate matter, organic pollutants, and physical factors can produce complex effects such as synergistic, antagonistic, or additive effects, involving multiple molecular pathways such as changes in absorption and metabolism, oxidative stress, mitochondrial dysfunction, and epigenetic regulation. In recent years, relevant studies have initially revealed some combined toxicity mechanisms, providing a basis for understanding the health effects of combined exposure. At the same time, it also suggests that abnormal molecular markers may become new targets for early risk warning. This review systematically summarizes the toxic effects and mechanisms of BaP co-exposure, aiming to offer new insights for preventing and treating BaP-related disorders.

Graphical abstract

Public health; Environmental health; Pollution; Environmental toxicology

Introduction

Benzo[a]pyrene (BaP) is one of the major toxic polycyclic aromatic hydrocarbons (PAHs) and is usually used as its representative. As a ubiquitous environmental pollutant, BaP derives from the incomplete combustion and pyrolysis of organic materials such as traffic or industrial emissions, fossil fuel combustion, cigarette smoke, and cooking oil fumes.¹ Despite world-spread awareness of its identified carcinogenicity to humans, exposure to BaP is almost unavoidable in daily life. For general people, tobacco smoke and polluted air are the main sources of BaP exposure. Both smokers and passive smokers inhale high concentrations of BaP, which are reported to range from 14 to 65 ng/cigarette.² BaP levels in ambient air worldwide have been reported to exceed its maximum permissible risk level of 1.0 ng/m³ stipulated in European legislation, especially in industrial cities.^3,4 Besides, dietary intake is another common source of BaP exposure for the general population, mainly including high-temperature processed food and vegetables grown in contaminated soils.^5,6 Therefore, people who are exposed to any other toxicant are likely to be co-exposed with low-dose BaP from the polluted living environment.

After entering the human body, BaP primarily undergoes a series of metabolic processes in the liver catalyzed by CYP1 enzymes and epoxide hydrolases to produce dihydroxy-epoxybenzo[a]pyrene and exert its toxicity, which is subsequently metabolized or excreted via ATP-binding cassette transport proteins.⁷ Currently, low doses of BaP can cause acute or chronic damage to the reproductive system, immune system, digestive system, and nervous system.^8,9,10,11 In addition, common air pollutants to which the general population is exposed, such as heavy metals, particulate matters (PMs), or volatile organic compounds (VOCs) or other pollutants in aerosols, often enter the human body in combination with BaP.¹² In recent years, some studies have focused on the damage effects on health caused by the co-exposure of BaP and other pollutants, and found that the effects of BaP on other toxins are two-sided. For example, BaP can exacerbate the immunotoxic effects of mercury¹³ and inhibit the biological antioxidant toxic effects of copper as well.¹⁴ Therefore, the different effects of BaP on other pollutants highlight the complex interaction patterns between environmental pollutants and the organism, which is worth further exploration.

Current research concerning the toxicological effects of BaP combined with other environmental toxicants remains scattered and simplistic. Most existing reviews and experimental studies primarily focus on the toxic damage induced by BaP alone, while a few investigations address the combined harm of BaP and ultraviolet radiation or arsenic (As). Moreover, most studies generally ignore the potential variability of the co-exposed effects on the human body under different concentrations, durations, and exposure conditions, which greatly restricts the in-depth understanding of combined toxicity mechanisms between BaP and various prevalent environmental pollutants. In this review, we focused on health hazards and the underlying mechanisms induced by the co-exposure to BaP and other common environmental pollutants, including metals and metalloids, PM, nanoparticles, microplastics, organic contaminants, as well as electromagnetic radiation and air pollution-related factors, aiming to provide novel insights for the risk assessment, prevention, and control of environmental pollutant-related diseases.

Search strategy and selection criteria

A retrospective review over the past 30 years (1996 to April 30, 2026) was conducted by searching PubMed, Web of Science, Medline, Scopus, Embase, and Google Scholar. The formal Boolean search string: BaP AND (coexposure OR combined exposure OR combined effect OR synergistic effect) was searched across title, abstract, and keywords. Reference lists of key reviews were manually traced for extra relevant studies. The studies included in this article are all in vitro or in vivo experiments (rodents, C. elegans, zebrafish). Epidemiological/clinical research, single-BaP exposure experiments, incomplete abstracts without full text, and non-English inaccessible papers were excluded. All retrieved citations were first imported into literature management software to remove duplicate entries. Two independent researchers sequentially conducted primary screening by titles and abstracts to exclude obviously unqualified literature according to predefined inclusion and exclusion criteria. Any discrepancy was settled by consensus discussion. Classic historical literature and systematic reviews containing pivotal raw experimental conclusions were additionally enrolled after full-text assessment.

Metals or metalloid compounds

Over the centuries, metals and metalloids or their compounds have caused quite serious pollution to the environment. Humans are inevitably co-exposed to BaP as well as metals/metalloids through airborne inhalation, waterborne contact, and dietary intake, which can accumulate persistently in organisms.¹⁵ In relevant experimental studies, common exposure routes for combined BaP and metals/metalloids treatment include drinking water administration, gavage, intratracheal instillation in model animals, and in vitro exposure in cell cultures. Studies have shown that their co-exposure has synergistic or antagonistic effects on the metabolic function, nervous system, carcinogenic progression, and reproductive system (Table 1).

Table 1.

Toxicity caused by co-exposure of BaP and metals or metalloids

System	Metals	Experimental subjects (route of exposure)	Dosage and exposure duration	Lesion location/health impairment (+/−/n)	References
ADME process	Nickel (NiCl2)/Cadmium (CdCl2)/Cobalt (CoCl2)	rats pretreated with metals (intratrecheal instillation),then the isolated lungs perfused with BaP	NiCl2(1M)/CdCl2 (1M)/CoCl2 (1M) + BaP (5 nM) for 45 min	BaP metabolism affected by NiCl2 (−), CdCl2 (+), CoCl2 (n)	Williams et al.16
	Cadmium (CdCl2)	adult zebrafish (water)	BaP (4.25 nM) + Cd (89.3 nM), 72 h	increased absorption rate of Cd and BaP (+)	Kodzhahinchev et al.17
	Cadmium (CdCl2)	adult zebrafish (water)	BaP (0.44 or 1.07 μg/L) + Cd (5.8 or 22 μg/L), 72 h	Cd and BaP body accumulation (+); oxidative stress (n)	Kodzhahinchev et al.18
	Cadmium (CdCl2)	adult zebrafish (water)	BaP + Cd (0.1 + 1, 0.1 + 10, 1 + 1, 1 + 10 μg/L), 28 days (one time on the first day)	inhibition of each other’s detoxification pathway (−) disruption of cardiac function (+)	Kodzhahinchev et al.19
Nervous system	Aluminum-maltolate	the primary cortical neurons of rats	BaP (10 μM)+ Al (50, 100, 400 μM), 72 h	increased neuronal cell apoptosis (+)	Jinzhu et al.20
	lead (lead acetate)	mice (gavage)	BaP (0.5 and 5 mg/kg, gavage) + Lead Acetate (5.4 and 54 mg/L in drinking water), 30 days (daily)	spatial learning and memory impairments (+) DNA damage (+) an increased of malondialdehyde and a decrease in superoxide dismutase and glutathione content (+)	Youbin et al.21
	Cadmium (CdCl2)	a developing zebrafish model (water)	BaP (3 μM) + Cd (0.1, 0.3 μM), from 5 to 120 h postfertilization	dark-induced locomotor stimulation in the larval motility test (−), activity in the adolescent novel tank test (−)	Stickler et al.22
Cancer	Arsenic (As)	rat/hamster/mice	review	respiratory exposure: elevated incidence of squamous cell carcinoma of the rat lung; total adenomatous lung tumor incidence in hamster oral exposure: multiplicity and burden of lung adenomas (+); 30% of mice in arsenic and BaP co-exposure group also developed lung adenocarcinomas.	Wang et al.23
	Arsenic (NaAsO2)	immortalized human bronchial epithelial BEAS-2B cells	BaP (2.5 μM) + NaAsO2 (1 μM) for 30 weeks	Increased expression of METTL3 and RNA m6A methylation; anchorage-independent growth, cancer stem cell characters and tumorigenesis	Wang et al.24
	Arsenic (NaAsO2)	MDA-MB-231 human breast cancer cells	BaP (1, 2.5 μM) + NaAsO2 (0.01, 0.1 μM) 24 h	Accelerated migratory rate of breast cancer cells (+)	Maleki et al.25
	Cadmium (CdCl2)	human breast cancer MCF-7 cells	BaP (1 μM) + Cd (1 μM), 48 h	Enhanced carcinogenicity and metastasizing activity of MCF-7 cells (+)	Jungnickel et al.26
	Titanium (TiO2) Iron (Fe2O3) Aluminum (Al2O3)	hamster (intratracheal instillation)	BaP and TiO2/Fe2O3/Al2O3 (0.2 mL, 3 mg) 15 weeks (weekly)	BaP and TiO2/Fe2O3: Induction of papillomas, squamous cell carcinomas, and a few adenomas and adenocarcinomas in throat, trachea, and lungs (+) BaP and Al2O3: Induction of laryngeal and tracheal papillomas (+)	Stenbäck et al.27
Reproductive system	Lead (PbCl2)	female mice (BaP, gavage; PbCl2, in drinking water)	BaP (10 mg/kg/day) + PbCl2 (1 g/L), 7–16 days	decline in fertility of oocytes (+)	Kristensen et al.28
Others	Cadmium (CdO)	mouse epidermal cells	BaP (0.05%–0.1% of culture volume) + CdO (below 25 μM), 16 h	death of epidermal cells (+); DNA damage (+)	Mukherjee et al.29

BaP, benzo[a]pyrene, ADME, abbreviation for absorption, distribution, metabolism and excretion, METTL3, methyltransferase-like 3. +, synergistic or additive effect; -, antagonistic effect; n, no significant interactive effects.

ADME process

Studies have found mutual interactions between BaP and metals/metalloids in terms of in vivo absorption and metabolism. S J Williams et al. intratracheally administered NiCl₂, CdCl₂, or CoCl₂ (1 mol) to rats, followed by arterial infusion of BaP (5 nmol) 45 min later, and found that the metabolism of BaP was slightly inhibited by NiCl₂, accelerated by CdCl₂, and unchanged after CoCl₂ exposure.¹⁶ In adult zebrafish, acute 72-h co-exposure to BaP and Cd significantly elevated the short-term uptake rate of both contaminants within 3–6 h. Specifically, Cd presented an increased maximum uptake rate in the gills without overexpression of gill transporters (epithelial calcium channels and divalent metal transporter 1), while BaP reached the highest whole-body uptake during the first 6–12 h.¹⁷ Likewise, co-exposure elevated tissue accumulation of Cd and BaP, accompanied by marked upregulation of cytochrome p4501a (CYP1A1) and metallothionein-2 (MT2), with no obvious alteration in oxidative stress status.¹⁸ Notably, distinct from acute toxicity, 28-day sub-chronic co-exposure to BaP and Cd did not change the body burden of Cd in zebrafish. The two contaminants mutually inhibited detoxification enzyme expression and disrupted cardiac function. In detail, BaP suppressed gill MT2, and Cd blocked the induction of CYP1A1 in both gills and liver tissues.¹⁹ Collectively, acute co-exposure to Cd and BaP promotes the absorption and bioaccumulation of both pollutants without triggering additive toxic effects. By comparison, sub-chronic co-exposure reshapes the profiles of key metabolic and detoxification enzymes, ultimately leading to physiological dysfunction. The interactive impacts of BaP and metals/metalloids on reciprocal ADME behaviors rely largely on metal types, tested organisms, as well as exposure dose and duration.

Nervous system

Many metals exert potent neurotoxicity,^30,31 while BaP could also induce apoptosis of nerve cells.³² Current studies on the neurological toxicity induced by their co-exposure mainly focus on aluminum (Al), lead (Pb), and cadmium (Cd) (Figure 1). Researchers have found that co-exposure of BaP and Al can target the central nervous system and exert synergistic effects on apoptosis in neurocytes in vitro,²⁰ which indicates that the long-term combined exposure is likely to exacerbate the damage to the central nervous system. Likewise, 30-day co-exposure of BaP and Pb exerted synergistic effects on spatial learning and memory impairment in mice as well as DNA damage. Meanwhile, their co-exposure produced additive effects on the increased level of malondialdehyde (MDA) and decreased levels of superoxide dismutase (SOD) and glutathione (GSH), suggesting that oxidative stress damage may be the underlying mechanism.²¹ Recently, in a developing zebrafish model, the effects of BaP and Cd on zebrafish behavior were largely independent of one another. Nevertheless, their binary mixture may produce additive neurobehavioral outcomes at specific developmental stages. For instance, BaP alleviated Cd-induced locomotor deficits under dark stimulation in larvae, while Cd attenuated BaP-induced hyperactivity in adolescent zebrafish.²²

Figure 1.

Neurological damage and carcinogenicity induced by BaP and metals/metalloids co-exposure

BaP, benzo[a]pyrene; Pb, lead; Al, aluminum; Cd, cadmium; As, arsenic; Ti, titanium; Fe, iron; BPDE, benzo[a]pyrene epoxide; CYP1A1, cytochrome p4501a; METTL3, methyltransferase-like 3; SUV39H1, histone methyltransferase. +, synergistic or additive effect; -, antagonistic effect.

Cancer

Numerous toxicological studies confirmed that multiple metallic and metalloid substances, including As, Cd, titanium (Ti), iron (Fe), Al, and plutonium (Pu), exerted joint tumor-promoting effects with BaP (Figure 1). Among them, the co-exposure of BaP and As can significantly increase the risk of lung cancer. Zhishan Wang comprehensively reviewed the carcinogenic risks of As and BaP co-exposure in 2021.²³ In rats, respiratory co-exposure increased the occurrence of lung squamous cell carcinoma and adenomatous tumors. Oral co-exposure elevates the multiplicity and burden of pulmonary adenomas, and develops into lung adenocarcinomas (approximately 30%). Mechanistically, genetic toxicity damage, epigenetic modifications, and activation of oncogenic pathways were involved. The combined exposure of As and BaP inhibited DNA damage repair, increased the level of BaP epoxide (BPDE)-DNA adducts and micronucleus formation, and modulated CYP1A1 activity. Such genotoxic outcomes are model-, dose-, and time-dependent, with contradictory or insignificant results frequently observed across diverse experimental conditions. Epigenetically, As and BaP co-activated aryl hydrocarbon receptors (AhRs) to upregulate histone methyltransferase SUV39H1 and then facilitated the dimethylation of H3 lysine 9, which implied that the epigenetic dysregulation involving SUV39H1 played an important role in the synergistic lung tumorigenesis. In terms of oncogenic signal transduction, the inhibition of SOCS1 expression activated the PI3K/Akt and ERK1/2 signaling pathways, thereby inducing the formation of tumor stem cell-like phenotypes. While thymocyte apoptosis induced by co-exposure led to immunosuppression and ultimately promoted tumor development.²³ Subsequently, in 2023, he identified epitranscriptomic dysregulation as a newly discovered synergistic mechanism in lung cancer caused by co-exposure to As and BaP. Both in vivo and vitro experiments have confirmed that the combined exposure upregulated the expression of METTL3 and the m6A methylation of overall RNA, thereby inducing cancer stem cell characteristics and tumor formation.²⁴ Another in vitro assay demonstrated that As and BaP co-exposure enhances cell viability and migration ability of breast cancer cells without inducing epithelial-mesenchymal transition (EMT).²⁵ Additional in vivo research is required to evaluate their combined effects on the progression of breast cancer.

Besides, the mixed exposure of BaP (1 μmol) and Cd (1 μmol) had a synergistic effect on the tumorigenic potency of human breast cancer MCF-7 cells.²⁶ The combined use of BaP with Ti dioxide or Fe oxide could induce papillomas and squamous cell carcinoma of the larynx, trachea, and lungs, as well as a few adenomas and adenocarcinomas, while BaP in combination with Al oxide could induce papillomas of the larynx and trachea in mice or rat.²⁷

Reproductive system and others

The study has shown that female mice exposed to BaP or Pb simultaneously have a significantly longer gestation period and more impaired fertility than those exposed to BaP or Pb alone.²⁸ In addition, co-exposure of BaP and Cd can also induce apoptosis in mouse epidermal cells due to the effect of Cd on the formation of BPDE-DNA adducts.²⁹

Collectively, co-exposure to BaP with various metals and metalloids interactively altered its ADME process, and further triggered neurotoxicity, carcinogenic risk, and reproductive dysfunction via oxidative stress, DNA damage, epigenetic, and epi-transcriptomic dysregulation. However, the mechanistic exploration remained fragmented, and the regulatory networks and interactions among genes, epigenetics, and signaling pathways during the combined exposure process remain unclear. Besides, the long-term developmental and reproductive toxicity from the combined exposure of BaP and metals/metalloids is largely unknown, and their effects on systemic toxicity responses, immune dysfunction, and multi-organ cumulative damage still require further investigation.

Particulate matters

PMs consists of solid and liquid particles suspended in the atmosphere derived from both natural and anthropogenic sources. Natural PM emissions primarily stem from volcanic eruptions and forest fires, whereas the anthropogenic PMs are mainly generated from industrial production, fuel combustion, and traffic emissions. This common environmental pollutant often attains relatively high concentrations in the atmosphere and readily adsorbs diverse hazardous substances, including metals, PAHs, and other persistent organic pollutants (POPs),³³ serving as an effective carrier for environmental toxicants. In this section, we have comprehensively summarized the published literature concerning the combined toxic effects induced by co-exposure to BaP and a broad spectrum of PMs, which were categorized into conventional atmospheric particulates, inorganic, and emerging organic particulates (Table 2). Specifically, conventional atmospheric particulates mainly refer to PM2.5 and PM10. Inorganic PMs cover carbon-based particles (carbon black (CB), multi-walled carbon nanotubes, and C60 fullerenes), silica nanoparticles (SNPs), mineral particles (asbestos and crystalline silica), and metallic nanoparticles. Emerging organic particulate contaminants primarily include micro-/nano-plastics, represented by polystyrene (PS) nanoparticles.

Table 2.

Toxicity caused by co-exposure of BaP and particulate matters

PM species	Contaminant	Experimental subjects (route of exposure)	Dosage and exposure duration	Lesion location/health impairment (+/−/n)	Reference
Inorganic carbon-based particles	carbon black (CB)	A549 cell line	BaP (50 mg) + CB (size 0.1, 2.5, 10 μm, dose 2 g) 24 h	the smaller the size of CB, the more ROS formation and cell damage (n)	Ryu et al.34
	CB	THP-1 cell line	CB particles coated with 10 μg BaP/g particles, 24 h	inhibition of IL-1β (+)	Goulaouic et al.35
	carbon (C)	Hamster (intravenous injection)	BaP and C dust (0.2 mL,3 mg) 15 weeks (weekly)	increased rate of papillomas of throat, trachea and lungs and lung adenocarcinoma (+)	Stenbäck et al.27
	ultrafine CB (P90)	PBMC cell line	BaP (0.1, 1, 10 μM) + P90 (32 μg/mL), 3 h	P90 decreases the activation or detoxification of inhaled BaP (−)	Eder et al.36
	multi-walled carbon nanotubes (MMCN)	pulmonary epithelial cells	BaP (10, 20, 40 and 80 μM) + MMCN (1, 2, 4, 8, 16, 32, 64, 128 and 256 μg/mL), 48 h	generation of ROS (+); apoptosis (+); oxidative DNA damage (+)	Azari et al.37
Fullerenes	C60	Zebrafish embryos (microinjection)	BaP (8 μg/L) + C60 (20 mg/L) 72 h	cell damage (+)	Della Torre et al.38
	C60	Adult zebrafish (water)	BaP (0.01,0.1 and 1 μg/L) + C60 (1 mg/L), 4 h	the intake of BaP (+); Cellular vitality, detoxification effect of intracellular enzymes (−)	Ferreira et al.39
Silica nanoparticles	SNPs	Zebrafish embryos (microinjection)	BaP (0.125 mg/mL) + SNPs (3 mg/mL) 24 h	pericardial effusion, bradycardia, and aggregation of red blood cells in the tail vein (+) toxicity to the immune system, inflammatory processes, and cardiovascular development (+)	Duan et al.40; Asweto et al.41
	SNPs	HUVECs	BaP (1 mM) + SNPs (10 μg/mL) 24 h	cell viability (−); intracellular ROS production (+); oxidative damage (+); DNA damage (+); cell-cycle arrest and apoptosis (+)	Asweto et al.42
Mineral particles	SiO2	Mice (intratrecheal instillation)	BaP + SiO2 (0.05 + 2.5, 0.1 + 2.5, 0.2 + 2.5 mg) Observed at 1, 2, 3, 7 days after a single administration	pneumonia and oxidative damage (+)	Tan et al.43
	short or medium chrysotile asbestos	Fibroblast (from the foreskin of a normal white male newborn)	BaP (0.1–10 μM) + short or medium chrysotile asbestos (100 μg/mL) 24 h	cell viability (n) for short chrysotile asbestos cell viability (+) for medium chrysotile asbestos	Stephens et al.44; Ahamed et al.45
	chrysotile asbestos	Monkey kidney cells	BaP + chrysotile asbestos (0.0039 μM + 0.1 mg, 0.039 μM + 0.5 mg), 24 h	the induction of interferon (−)	Hahon et al.46; Deval et al.47
	crocidolite	hamster (tracheal epithelial cells)	BaP-coated crocidotile asbestos (2 × 10−7mol BaP on 3 μg chrysotile or 10 μg crocidolite/mL medium), 1, 2, 4 or 8 h	Carcinogenesis of cells (+)	Mossman et al.48; Ahamed et al.49
Metallic nanoparticles	bismuth oxide nanoparticles (BO NPs)	GC-1 mouse spermatogonial cells	BaP (20 μM) + BONPs (40 μg/mL) 24 h	cell viability (−); cytotoxicity (+)	Ahamed et al.45; Ren et al.50
	cerium dioxide nanoparticles (CeO2 NPs)	chorion and primary cytotrophoblasts (from pregnant full-term human placenta)	BaP (0.1, 0.6 or 1 μM) + CeO2 NP (40 or 64 μg/mL) 30 min, 4 h or 24 h	DNA damage (+); modulation of the genotoxic effect of BaP (−)	Deval et al.47
	Nickel (II) oxide nanoparticles (NiO NPs)	HepG2 human hepatoma cells Primary rat hepatocytes.	BaP (10 μM) + NiO NPs (25 μg/mL) 24 h	Cytotoxicity (+); increased oxidative stress response and apoptosis (+)	Ahamed et al.49
Organic microplastics/nanoplastics	Polystyrene nanoparticles (PS NPs)	Caenorhabditis elegans	BaP (20 μg/L) + PS NPs (10 μg/L)	shortened lifespan and multiple aging phenotypes (+)	Stephens et al.44; Ren50
	PS	Mice (gavage)	PS @ BaP (0.25 mg/kg, daily) 28 days	colonic barrier degradation, body weight loss, colon length shortening, oxidative stress, autophagy, inflammation, and bacterial translocation (+)	Hahon et al.46; Shaoyong et al.51
	PS	male Kunming mice (drinking water) HK-2 and HIEC-6 cell line	PS @ BaP mixture (400 μg/mL) 56 days cell culture (200 μg/mL mixture)	disrupted lipid metabolism and ferroptosis in the kidneys (+) HK-2: the above effects were not observed. HIEC-6: ferroptosis (+)	Mossman et al.48; Zhang et al.52

BaP, benzo[a]pyrene; PM, particulate matter; C₆₀, fullerene nanoparticles; SNPs, silica nanoparticles; SiO₂, micron-sized crystalline silica particles; PS, polystyrene; ROS, reactive oxygen species; HK-2, human renal tubular epithelial cells; HIEC-6, human small intestinal epithelial cells; A549, human non-small cell lung cancer cells; PBMC, primary human peripheral blood mononuclear cells; THP-1, human monocytic leukemia cells; HUVECs, human umbilical vein endothelial cells. +, synergistic or additive effect; −, antagonistic effect; n, no significant interactive effects.

Atmospheric particulates and carbon-based particles

Massive researches have shown that BaP is one of the most hazardous components of conventional atmospheric particulates (e.g., PM2.5 and PM10), and higher concentrations of PM2.5-associated BaP were detected during cold seasons relative to warm seasons,⁵³ which was mainly attributed to increased pollutant emissions from gasoline and diesel vehicles alongside additional heating-related pollution sources in cold weather.⁵⁴ However, there are few toxicological studies on the combined exposure. An experiment in mice showed that urban dust PMs plus BaP resulted in increased DNA strand breaks by the inhibition of CYP1A1and CYP1B1 bioactivation.⁵⁵

CB, an important industrial material and a prevalent constituent of atmospheric PMs, has been classified as a Group 2B carcinogen. Co-exposure to CB and BaP frequently occurs in occupational populations, such as firefighters.⁵⁶ A number of experimental studies have revealed the adverse toxic effects induced by their co-exposure (Figure 2). In human A549 lung cell models exposed to different sizes of BaP-loaded CB particles (0.1, 2.5, and 10 μm), the toxicity was inversely correlated with particle size instead of BaP presence. Smaller CB particles reduced cell viability and promoted reactive oxygen species (ROSs) and IL-6/8 formation.³⁴ While BaP coating on CB (14 nm) amplified the inhibitory effect of CB against IL-1β secretion but did not modify IL-8 formation in THP-1 macrophages,³⁵ suggesting that such interactive effects relied on both particle size and cell type. In contrast, BaP combined with C induced papillomas of the throat, trachea, and lungs, as well as lung adenocarcinoma in hamster.²⁷ Consistently, ultrafine CB (P90) inhibited the expression of the CYP1B1 gene in human monocytes, which impaired the cellular activation and detoxification capacity of BaP.³⁶ Furthermore, a stronger cytotoxicity (ROS, oxidative DNA damage, and apoptosis) in human lung epithelium was observed when co-exposed to BaP and multi-walled carbon nanotubes,³⁷ which might be attributed to their large surface area and the ability to absorb intrinsic toxic substances such as PAHs.⁵⁷

Figure 2.

Toxicity induced by co-exposure to BaP and inorganic carbon-based particles

BaP, benzo[a]pyrene; CB, carbon black; P90, ultrafine carbon black; MWCNTs, multi-walled carbon nanotubes; C, carbon; C₆₀, fullerene nanoparticles; ROS, reactive oxygen species; IL, interleukin; CYP, cytochrome p450; 8-OHdG, 8-Hydroxy-2′-deoxyguanosine; +, synergistic or additive effect; -, antagonistic effect; n, no significant interactive effects. Arrows indicate promotion, and blunt-ended lines represent inhibition.

Fullerene nanoparticles are stable carbon-only nanomaterials with diameters ranging from 30 to 150 nm under environmental conditions. The booming application of nanomaterials in pharmaceutical, energy, and electronic industries has rendered fullerene nanoparticles ubiquitous in diverse environmental media.⁵⁸ Previous toxicological studies demonstrated distinct stage-dependent toxic interactions between BaP and fullerene nanoparticles (C₆₀) in zebrafish models (Figure 2). When zebrafish embryos were simultaneously exposed to BaP and fullerene nanoparticles, the bioavailability and intake of the nanoparticles by embryos decreased, along with reduced oxidative stress, and proteomic analysis found an increased number of energy metabolism-related proteins in embryos.³⁸ On the contrary, in adult zebrafish, fullerene nanoparticles enhanced the cellular uptake of BaP and diminished the level of ROS induced by BaP exposure, accompanied by decreased cell viability and a weakened detoxification effect of phase Ⅱ enzymes such as Glutathione S-transferase.³⁹

Silica and asbestos particles

SNPs are defined as amorphous silicon dioxide particles with primary particle sizes ranging from 10 to 100 nm. And its widespread industrial utilization leads to frequent environmental release of SNPs, resulting in potential health hazards to humans when co-exposed to BaP (Figure 3). Co-exposure of zebrafish embryos to BaP and SNPs can induce pericardial edema and bradycardia, as well as the aggregation of red blood cells in the embryonic tail vein. These effects may be attributed to the enhanced expressions of pro-inflammatory and pro-coagulant genes, along with markedly increased levels of tissue factor and phosphorylated AP-1/c-Jun.⁴⁰ Similarly, another study also found that low-dose co-exposure of BaP (0.125 mg/mL) and SNPs (3 mg/mL) had a stronger toxic effect on the immune system, inflammatory processes, and cardiovascular development of zebrafish embryos than the individual exposure.⁴¹ The MAPK signaling pathway, calcium signaling pathway, p53 signaling pathway, PI3k/Akt signaling pathway, and several pathways associated with immune response were the most prominent pathways induced by co-exposure of SiNPs and B[a]P in zebrafish embryos using whole genome microarrays.⁴¹ In vitro, simultaneous exposure to BaP and SNPs could produce a synergistic effect on ROS production, leading to cell-cycle arrest and apoptosis in human umbilical vein endothelial cells.⁴² Besides, acute intratracheal instillation of BaP and micron-sized crystalline silica particles in mice caused more severe pulmonary inflammation and oxidative damage when compared with individual exposures.⁴³

Figure 3.

Toxicity induced by co-exposure to BaP and silica/asbestos particles

BaP, benzo[a]pyrene; SNPs, silica nanoparticles; TF, tissue factor; ROS, reactive oxygen species; HUVECs, human umbilical vein endothelial cells. +, synergistic or additive effect; Arrows indicate promotion.

Asbestos has been classified as a Group 1 human carcinogen by IARC, which can induce diffuse pulmonary fibrosis and even malignant tumors, including lung cancer and pleural mesothelioma.⁵⁹ Evidence has shown that cigarette smoking, a major source of BaP, exerts synergistic effects on asbestos-induced lung cancer risk, and asbestos does not promote the formation of BaP-DNA adducts. However, the survival rate of human normal fibroblasts co-exposed to BaP and intermediate chrysotile asbestos in vitro is higher than that exposed to chrysotile asbestos alone, indicating more severe pulmonary fibrosis under combined exposure.⁴⁴ Moreover, co-treatment with activated BaP and thermal asbestos produced stronger inhibitory effects on interferon induction in rhesus monkey kidney cells than either individual exposure alone,⁴⁶ which suggested that bio-activated BaP and asbestos co-inhibited the biological defense mechanism. Additionally, thermal asbestos can also affect the ADME process of BaP. Under serum-free conditions, co-exposure to BaP and thermal asbestos significantly promoted cellular BaP uptake and metabolism, as well as the binding efficiency of BaP to intracellular nucleic acids in normal human fibroblasts.⁶⁰ Consistently, BaP was found to be retained in hamster tracheal cells 8 h post-exposure when exposed to BaP-coated chrysotile asbestos.⁴⁸ Collectively, the combined toxicity of asbestos and BaP depended primarily on asbestos fiber type. Asbestos facilitated cellular absorption and metabolic processing of BaP without directly altering BaP-DNA adduct formation, whereas BaP amplified the fibrogenic potential of asbestos (Figure 3).

Metal nanoparticles

Metal nanoparticles are ubiquitous emerging inorganic pollutants, which are widely applied in daily cosmetics, antibacterial products, electronic devices, and industrial materials, and continuously leak into environmental media. Several metallic nanoparticles have been confirmed to synergistically aggravate BaP-induced cytotoxicity predominantly via oxidative stress and cellular apoptosis. Bismuth oxide nanoparticles aggravated BaP-triggered cytotoxicity and decreased cell viability in mouse spermatogonia, resulting in reduced cell survival. Mechanistically, the loss of mitochondrial membrane potential induced by co-exposure in spermatogonia may lead to an increase in free oxygen radicals, including ROS and hydrogen peroxide, and a depletion of antioxidants, including glutathione and various antioxidant enzymes, thereby inducing excessive cellular oxidative stress.⁴⁵ Similarly, nickel oxide nanoparticles exerted synergistic hepatotoxicity with BaP in both HepG2 cells and primary rat hepatocytes through free oxygen radicals induced oxidative stress, revealed by lactate dehydrogenase (LDH) leakage, lipid peroxidation, mitochondrial membrane potential depletion, and apoptosis.⁴⁹ In contrast, cerium dioxide nanoparticles induced adverse placental cellular dysfunction independent of excessive oxidative injury, which bio-activated BaP to trigger DNA damage with an increased γ-H2AX level, and disturb the stress signaling in human trophoblasts by stabilizing the stress transcription factor p53 and inducing the expression of its target protein p21.⁴⁷

Emerging organic particles

Microplastics and nanoplastics, characterized by small particle sizes, strong mobility, and non-degradability, distribute ubiquitously in multi-environmental media. Their large specific surface areas allow efficient adsorption of BaP, resulting in prevalent co-exposure scenarios and potential synergistic toxic hazards to humans. Toxicological studies have validated the multiple organ toxicities induced by combined exposure to BaP and PS micro/nanoplastics. In Caenorhabditis elegans, co-exposure to BaP and PS nanoparticles impeded the removal of accumulated ROS, triggering mitochondrial dysfunction and significantly shortened lifespan, thus leading to accelerated organismal aging.⁵⁰ Similarly, BaP aggravated PS microplastic-induced colonic barrier damage by activating the Notch signaling pathway, which elevated intracellular ROS accumulation and increased intestinal permeability.⁵¹ Interestingly, a recent study has shown that PS and BaP can cause kidney damage in mice through the gut-kidney axis. Multi-omics analyses, including RNA sequencing, untargeted metabolomics, and 16S rRNA sequencing, further elucidated the intestinal and renal toxic mechanisms underlying PS microplastic and BaP co-exposure. Combined pollutant exposure suppressed the expression of intestinal tight junction proteins (ZO-1, claudin-1, and occludin), disrupting intestinal vascular barrier integrity and altering microbial community abundance. Such intestinal dysfunction led to an increase in intestinal metabolites, particularly polyunsaturated fatty acids, which translocated to the kidney via the gut-kidney axis. The accumulated metabolites further activated the renal ACSL4/LPCAT3 signaling axis, ultimately triggering renal ferroptosis and kidney damage.⁵²

Based on the current published literature, studies on the combined exposure of common and emerging PMs and BaP are still limited and insufficiently comprehensive. Moreover, current mechanistic explorations remain superficial. Besides, there is a scarcity of studies focusing on long-term, low-dose co-exposure scenarios that closely mimic real human exposure conditions, resulting in inadequate evidence for carcinogenicity, developmental toxicity, and long-term adverse health outcomes. Moreover, current mechanistic explorations remain superficial. Integrated multi-omics approaches (transcriptomics, proteomics, metabolomics, and epigenetics) are rarely applied to systematically decipher the molecular regulatory networks underlying co-exposure-induced damage.

Organic pollutants

Organic pollutants, a class of carbon-based chemicals, are predominantly generated from anthropogenic activities, including industrial production, agricultural pesticides, incomplete combustion processes, and vehicle emissions. Organic pollutants are taxonomically diverse toxic contaminants. Long-term exposure to some of them has been reported to cause carcinogenicity, endocrine disruption, neurotoxicity, and immune suppression in humans. Notably, BaP frequently coexists with various organic pollutants in real environmental scenarios, resulting in complex combined biological effects. In this section (Table 3), we systematically summarized the existing toxicological studies on co-exposure to BaP and diverse organic pollutants (endocrine disruptors, VOCs, tobacco alkaloids, and food-processing-derived pollutants), aiming to elaborate their interactive toxic behaviors and underlying joint pathogenic mechanisms.

Table 3.

Toxicity caused by co-exposure of BaP and organic pollutants

Type	Contaminant	Experimental subjects (route of exposure)	Dosage and exposure duration	Lesion location/health impairment (+/−/n)	References
Endocrine disruptors	3,3′-dichlorobiphenyl sulfate (PCB11)	Zebrafish embryos	BaP+ PCB11 (100 μg/L+20 μM for 1–4 days, 100 μg/L+0.2 μM for 1–15 days)	activity of BaP (−); Hepatic lipid accumulation (+)	Roy et al.61
	2,2′,4,4',5,5′-hexachlorobiphenyl (PCB153)	HepG2 human hepatoma cells	Pretreated with PCB153 for 48 h, then treated with BaP (50 μM)+ PCB126 (0.1, 1, 10, 100 μM) for another 24 h	enhanced formation of micronuclei and activity of cytochrome P450 (+), decreased activity of glutathione S-transferase (−)	Wei et al.62
	3,3′,4,4',5-pentachlorobiphenyl (PCB126)	HepG2 cells	Pretreated with PCB126 for 48 h, then treated with BaP (50 μM)+ PCB126 (0.01, 0.1, 1, 10 nM) for another 24 h	increased oxidative stress and impaired control of cell cycle (+)	Wei et al.63
	Perfluorooctane sulfonate (PFOS)	Rat C6 glioma cells	PFOS: EC50, 1.88 mg/L, BaP: EC50, 3.36 mg/L BaP + PFOS: 1/16, 1/8, 1/4 or 1/2 EC50, 48 h	increased oxidative stress and early apotosis (+); steroid hormone synthesis disorder through the activation of CYP1A1, Hsd17b8 and Dhcr24/7 (+)	Lu et al.64
	Dibutyl phthalate (DBP)	Rats (gavage)	BaP + DBP (1 + 50, 5 + 250 mg/kg), 90 days	pathological inflammatory changes and abnormal function of the liver (+)	Chen et al.65; Chen et al.66
	DBP	L02 human normal hepatocytes	BaP + DBP (12.5 + 25, 25 + 50, 50 + 100 μM), 72 h	apoptosis of liver cells (+); inflammatory factor secretion and hepatocyte damage (+)	Liu et al.67
	DBP	Rats (gavage)	BaP (1 mg/kg) + DBP (50 mg/kg), 90 days	spleen injury and secretion of pro-inflammatory cytokines (+)	You et al.68
	DBP	Rats (gavage) NRK-52E and NRK- 49F cells	Rats: BaP (1 mg/kg) + DBP (50 mg/kg), 90 days In vitro: BaP + DBP (2.5 + 5, 5 + 10, 10 + 20, 20 + 40, 40 + 80 μM), 72 h	renal inflammation, excessive extracellular matrix and fibrosis (+)	Chen et al.69
	Chlorpyrifos	PC12 cells	BaP (1, 10 μM) + Nicotine (30 μM), 6 days	suppressed cell growth (+); phenotype shift toward the dopaminergic cells (+)	Slotkin et al.70
	Sudan I	Rats	BaP (150 mg/kg, gavage) + Sudan I (50 mg/kg, intraperitoneal injection) 3 days	BaP-DNA adduct levels and the genotoxicity of BaP (+)	Dračínská et al.71
	1- or 2-naphthylamine (NA), 2-, 3-, or 4-aminobiphenyl (ABP)	RT-4 human urinary bladder papilloma cells	BaP (1 μM) + 1- or 2-NA or 2-,3- or 4-ABP (0.1, 0.3, 1, 3, 10, 30, 100 μM), 24 h	BaP-induced CYP1A1 activity was modulated by 1- or 2-NA (+), and 2-, 3- or 4-ABP (−)	Plöttner et al.72
Volatile organic compounds	Ethanol	Steatosis models (HepaRG and WIF-B9 cell lines) and obese zebrafish larvae	HepaRG (BaP, 1 or 2.5 μM + ethanol, 25 mM), 15 days WIF-B9 (BaP, 10 nM + ethanol, 5 mM), 5 days Zebrafish (BaP, 25 nM + ethanol, 43 mM), 7 days	contribute to the progress of a steatohepatitis-like state (+) increased levels of heme and hemin (+) in zebrafish larvae.	Bucher et al.73; Imran et al.74
	Ethanol	Steatotic HepaRG cells	BaP (2.5 μM) + Ethanol (25 nM) 2 weeks	decreased respiratory chain activity and DNA levels of cellular mitochondria (+) production of ROS (+)	Bucher et al.75
	Ethanol	WIF-B9 hepatocytes	BaP (10 nM) + Ethanol (5 mM), 5 days	cell death of fatty liver cell (+)	Tête et al.76
	Ethanol	Primary rat hepatocytes	BaP (100 nM) for 18 h before ethanol (50 mM) for 5 or 8 h	cell death (+)	Collin et al.77
	Ethanol	Male rats (gavage)	BaP (100 μg/kg) on alternative days + Ethanol (2 g/kg) daily 60 days	male reproductive toxicity (+); Androgen deficiency (+); increased oxidative stress (+)	Reddy et al.78
	Phenol	Rats (tracheal perfusion)	BaP (5 mg/monthly) + Phenol (0.4 mg/m3) 3 months	disruption of energy exchange in the lungs, liver, and kidneys (+)	Skvortsova et al.79
	O-cresol	CC57Br female mice (gavage)	BaP (1 or 5 mg) + O-cresol (0.02, 1, or 10 mg) 26 to 30 weeks	BaP modifies o-cresol induced carcinogenesis of forestomach tumors depends on the dose and the sequence of administration. BaP (1 mg) + o-cresol (1 mg) simultaneously, carcinogenesis (+); BaP before or after o-cresol, BaP (5 mg) + o-cresol (10 mg), carcinogenesis (−).	Yanysheva et al.80
Alkaloids/tobacco pollutants	Nicotine	GH rat pituitary tumor cells	BaP + Nicotine (5 + 0.1, 5 + 0.5, 5 + 10, 5 + 50 μg/mL)	inhibits cell growth and hormone synthesis (−)	Chakrabarti et al.81
	Nicotine	PC12 cells	BaP (1, 10 μM) + Nicotine (10 μM), 6 days	cell growth (n); suppression of differentiation into both dopaminergic and cholinergic phenotypes (+)	Slotkin et al.70
	Nicotine	pregnant rats (subcutaneous minipump)	BaP (30 mg/kg/day) + Nicotine (2 mg/kg/day), throughout gestation and into the first postnatal week	developmental Neurotoxicity (+) deficit in the development of acetylcholine and 5-hydroxytryptamine systems (+)	Slotkin et al.82
Food processing contaminants	2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)	mammary gland cells (MCF10A, CF12A)	BaP (100 pM) + PhIP (10 nM), 24 h	carcinogenesis of breast cell (+); reinforced ROS and ERK signaling (+); activation DNA oxidation and damage (+)	Pluchino et al.83
	Glycidamide (GA), Acrylamide (AC), PhIP	MCL-5 human B lymphoblastoid cells	A matrix of low-dose concentrations of BaP + GA/AC/PhIP	mutant frequency: low concentration (+), high concentration (−)	David et al.84; David et al.85
	N-nitroso compounds (NOCs)	A/J Min/+ mice (dietary intake)	BaP low (0.08) + NOCs (NDMA 0.4 + NPYR 0.6 + NPIP 0.01 + NDEA 0.02 + NTCA 80 + NMTCA 4.5 mg/kg bw/day) BaP high (8.00) + NOCs (NDMA 0.004 + NPYR 0.006 + NPIP 0.0001 + NDEA 0.0002 + NTCA 0.8 + NMTCA 0.045 mg/kg bw/day), 9 weeks	increased sex-dependent initiation of colorectal cancer in NOCs + BaP high dose group (+)	Johanson et al.86

BaP, benzo[a]pyrene; NRK-52E, normal rat kidney tubule epithelial cells; NRK- 49F, rat kidney interstitial fibroblast cells; PC12, rat adrenal medulla pheochromocytoma cells; CYP1A1, cytochrome P450 family 1 subfamily A member 1; EC₅₀, half-maximum effect concentration; NDMA, N-Nitrosodimethylamine; NPYR, N-Nitrosopyrrolidine; NPIP, N-Nitrosopiperidine; NDEA, N-nitrosodiethylamine; NTCA, N-Nitroso-2-methylthiazolidine 4-carboxylic acid; NMTCA, N-nitroso-2-methylthiazolidine 4-carboxylic acid. +, synergistic or additive effect; -, antagonistic effect; n: no significant interactive effects.

Endocrine disruptors

Endocrine-disrupting chemicals (EDCs) are exogenous substances that interfere with the synthesis, secretion, transport, metabolism, and binding of endogenous hormones, thereby disrupting the endocrine homeostasis of humans and wildlife, typically including POPs, heavy metals, bisphenols, phthalates, per- and polyfluoroalkyl substances (PFASs) and PAHs. Existing studies have investigated the metabolic process and genotoxicity induced by POPs and BaP co-exposure (Figure 4). Acute mixed exposure to BaP and 3,3′-dichlorobiphenyl sulfate (PCB11, 20 μM) significantly reduced the CYP1A1 activity and suppressed the metabolism of BaP in zebrafish embryos, while the chronic co-exposure increased hepatic neutral lipid accumulation in larvae.⁶¹ The opposite result was observed when both BaP and 2,2′,4,4',5,5′-hexachlorobiphenyl (PCB153) were exposed in human hepatoma cells. BaP in combination with PCB153 significantly enhanced the activity of CYP1A1 and the formation of micronuclei, but reduced the activity of Glutathione-S-transferase, indicating that PCB153 might increase BaP-induced genotoxicity.⁶² They further revealed an increased level of ROS and MDA and decreased percentage of cells in G0/G1 phase after co-exposure of BaP and 3,3′,4,4',5-pentachlorobiphenyl (PCB126) in HepG2 cells.⁶³ In addition, low-dose perfluorooctane sulfonate (PFOS) -BaP binary mixture induced neurotoxicity manifested as excessive oxidative stress and early apoptosis in C6 rat glimo cells, and the combined effect changed from additive to synergistic from 0.33 to 1.31 mg/L. Combined with targeted metabolomics analysis, binary exposure to PFOS and BaP activated the biosynthesis pathways of neurotransmitter-related phenylalanine, tyrosine, and tryptophan regulated by CYP1A1, Hsd17b8, Dhrc24, and Dhrc7, thereby inducing disturbance in steroid hormone synthesis.⁶⁴

Figure 4.

Toxicity induced by co-exposure to BaP and endocrine-disrupting chemicals (EDCs)

BaP, benzo[a]pyrene; PCB11, 3,3′-dichlorobiphenyl sulfate; PCB153, 2,2′,4,4',5,5′-hexachlorobiphenyl; PCB126, 3,3′,4,4',5-pentachlorobiphenyl; PFOS, perfluorooctane sulfonate; DBP, dibutyl phthalate; NA, ABP, . +, synergistic or additive effect; -, antagonistic effect. Arrows indicate promotion, and blunt-ended lines represent inhibition.

Dibutyl phthalate (DBP), one of the most commonly used phthalate esters, is widely applied as a plasticizer and additive, leading to human exposure through contaminated food and drinking water. Accumulating evidence has demonstrated that simultaneous exposure to BaP and DBP induces multi-organ toxicity in sub-chronic rodent models and in vitro experiments (Figure 4). In liver, combined BaP and DBP exposure triggered inflammatory injury and hepatic functional disorders. Mechanistically, the downregulated expression of miR-34a-5p activated the TLR4/MyD88/NF-κB cascade and facilitated Notch signaling-mediated M1 macrophage polarization, and synergistically reduced miR-122-5p expression, which regulated the SOCS1/STAT3 signaling pathway and further promoted hepatocyte apoptosis.^65,66,67 In the spleen, BaP and DBP mixture exacerbated pro-inflammatory cytokine secretion and tissue injury by inducing pyroptosis-dependent M1 macrophage polarization.⁶⁸ Additionally, co-exposure to BaP and DBP aggravated renal inflammation and fibrosis via the activation of fibroblast growth factor 23 (FGF-23)-regulated renin-angiotensin system (RAS) signaling in rats.⁶⁹ Co-exposure of BaP with other endocrine disrupters, such as pesticides, produces neurocytotoxicity. Chlorpyrifos aggravated BaP-induced suppression of cell growth and promoted BaP-induced phenotypic shift toward dopaminergic cells during the neurodifferentiation of PC12 cells.⁷⁰

Aromatic amines are ubiquitous semi-volatile EDCs and carcinogenic contaminants, which are primarily generated from the reductive degradation of widely used azo dyes (e.g., Sudan I) under anaerobic environmental conditions. An experiment revealed that when rats were exposed to BaP and Sudan I (benzidine-2-naphthol) in combination, the level of BaP-DNA adducts increased, thereby enhancing the genotoxicity of BaP.⁷¹ Typical aromatic pollutants, including 1- or 2-nitrophenol (NA), and 2-, 3-, or 4-aminoanisole (ABP), served as critical regulators of BaP-induced CYP1A1 bioactivity and exerted different directions of regulatory synergistic or antagonistic toxic effects in RT4 human bladder papilloma cells. Specifically, NA synergistically enhanced and ABP inhibited the activity and protein expression of CYP1A1 induced by BaP, and both showed an aromatic amine concentration-dependent relationship.⁷²

Volatile organic compounds

VOCs are a large group of organic compounds with high vapor pressure and low boiling point at room temperature, mainly including phenolic compounds, aldehydes, and aldehydes, ketones, alcohols, halogenated hydrocarbons, and esters. Ethanol, an important industrial raw material and the main component of alcoholic beverages, is considered to be harmful to health at any dose of alcohol consumption. Existing evidence has validated the synergistic hepatotoxicity and male reproductive toxicity induced by combined exposure to BaP and ethanol. Co-exposure to BaP and ethanol facilitated the pathological transition from simple hepatic steatosis to severe steatohepatitis in steatosis models.^73,74 In vitro assays using steatotic HepaRG cells revealed that BaP-ethanol co-exposure induces a steatohepatitis-like phenotype via impairing mitochondrial respiratory chain activity, reducing mitochondrial DNA content, and ROS overproduction.⁷⁵ Moreover, such synergistic cytotoxicity can remodel hepatic plasma membrane properties, ultimately resulting in the cell death of lipid-laden hepatocytes and primary rat hepatocytes.^76,77 Consistently, in high-fat diet-fed zebrafish model, BaP and ethanol induced elevated heme and hemin levels and mitochondrial Fe content, which led to mitochondrial dysfunction and thereby accelerated the pathological progression of non-alcoholic fatty liver disease (NAFLD).⁷⁴ Besides, 60-day repeated co-exposure to BaP and ethanol significantly reduces sperm quality, serum testosterone levels in rats, and increased fragmented DNA level in sperm, which might be due to the accumulated superoxide anion, hydrogen peroxide, and lipid peroxides in testes and epididymis and suppressed endogenous antioxidant systems.⁷⁸

Phenolic compounds represent an important category of VOCs, among which the simplest phenol has received particular attention as a potential carcinogenic activator. When rats were co-exposed to BaP and phenol for 3 months, it resulted in more severe disruption to energy exchange in the lungs, liver, and kidneys.⁷⁹ Furthermore, the carcinogenesis of BaP and o-cresol was dose-dependent and administration-sequence-dependent. Simultaneous exposure to equivalent doses (1 mg) of BaP and o-cresol significantly increased the incidence, multiplicity, and malignancy of forestomach tumors in mice. In contrast, o-Cresol administration before or after BaP at the same dose level (1 mg) or at super toxic doses (10 mg) of o-cresol with the BaP (5 mg) exerted inhibitory effects on carcinogenesis.⁸⁰

Tobacco pollutants

Nicotine is one of the alkaloids present in cigarette smoke, which is highly toxic and significantly addictive. Cigarette smoke is also an important source of BaP, and their co-exposure is inevitable.⁸⁷ S. Chakrabarti et al. found that the non-effective dose of nicotine treatment was able to enhance the inhibitory effect of BaP on cell growth and the synthesis of oxytocin and hormones in cultured rat pituitary tumor cells.⁸¹ Furthermore, co-exposure to nicotine and BaP induced combined neurotoxicity in experimental models. Nicotine strengthened BaP-induced inhibitory effects on the neural differentiation of PC12 cells into dopaminergic and cholinergic phenotypes. The developmental neurotoxicity caused by the combination of nicotine and BaP was disclosed in vivo.⁷⁰ When pregnant rats were treated with nicotine and BaP from early gestation to the first postpartum week, the offspring exhibited severe defects in presynaptic acetylcholine activity during adolescence and adulthood, accompanied by the dysregulated expression (upregulation or deficiency) of nicotinic acetylcholine receptors and serotonin receptors.⁸² These findings indicated that environmental nicotine exposure, including both active smoking and second-hand exposure, may have adverse effects on the neural development of fetuses and infants.

Food processing contaminants

High-temperature food processing generates various hazardous chemicals, including PAHs, heterocyclic amines (HCAs), acrylamide, and N-nitrosamines. Cumulative exposure to 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine and BaP contributed to enhanced initiation of breast cell carcinogenesis and led to enhanced acquisition of cancer-associated properties, such as increased DNA oxidation and damage, in which reinforced ROS elevation and ERK activation played an essential role.⁸³ Low-concentration co-exposure to a binary mixture of BaP and PhIP, glycidamide or acrylamide, which failed to induce mutagenesis under individual chemical treatment, significantly elevated mutation frequency (MF) in human MCL-5 cells. By contrast, high-concentration combined exposures reduced MF relative to single chemical exposure.^84,85 Both ethoxyresorufin-O-deethylase (EROD) activity and CYP1A1 mRNA levels were correlated with thymidine kinase MF, and G2/M phase cell-cycle arrest was observed following high-dose BaP and PhIP combined treatment, which corresponded to cellular DNA damage sensing and repair processes.⁸⁵ Additionally, high-dose co-exposure to BaP and NOCs induced sex-dependent initiation of colorectal cancer in A/J Min/+ mice.⁸⁶

In summary, existing toxicological evidence demonstrated that BaP consistently interacted with organic pollutants to induce divergent combined toxic responses and triggered multi-organ damage encompassing hepatic, renal, splenic, neuronal, and reproductive toxicity, as well as genotoxicity, carcinogenic initiation, and developmental neurotoxicity. Mechanistically, the disruption of endocrine homeostasis, impaired mitochondrial function, overproduction of oxidative stress and inflammation, dysregulated xenobiotic-metabolizing enzymes (mainly CYP1A1), and cell cycle disturbance collectively contributed to aggravated pathological damage and malignant transformation. Notably, the interactive toxic outcomes were highly dependent on pollutant subtypes, exposure dosage, administration sequence, and biological gender, resulting in complex and inconsistent joint toxic behaviors across different pollutant combinations and experimental models. Besides, sex-dependent differential toxicity and persistent intergenerational developmental hazards induced by BaP- organic pollutants mixture exposure received limited attention.

Electromagnetic radiation or air pollution indicators

Electromagnetic radiation

Electromagnetic radiation is found everywhere on Earth, which can be divided into high-energy ionizing radiation (e.g., X-rays, gamma rays, radioactive particles) and low-energy non-ionizing radiation (e.g., radio waves, visible light, microwaves). The health risks posed by electromagnetic radiation are primarily determined by its energy intensity. Ionizing radiation has been well documented to directly induce cellular DNA damage and elevate cancer susceptibility. However, the potential adverse health outcomes associated with long-term low-dose non-ionizing radiation exposure also need to draw public attention.

X-rays are commonly used in medical imaging, diagnosis, and industrial flaw detection. In cellular experiments using hamster embryonic cells, treatment with X-ray irradiation followed by BaP exposure demonstrated that X-rays enhanced BaP-induced cell transformation in a time-dependent manner. The maximal transformation rate was observed when BaP was supplemented 48 h post-irradiation, whereas this enhancing effect was significantly weakened at 72 h after radiation treatment.⁸⁸ In addition, BaP can also promote the carcinogenic potential of radioactive contaminants. When rats inhaled Pu oxide (220, 630, 6300 Bq, initial lung burden) for two to three weeks, the incidence of lung tumors increased, and survival rates decreased after intratracheal instillation of 0.2 mL of saline containing BaP (2 × 5 mg), confirming the synergistic carcinogenic interaction between radioactive elements and BaP.⁸⁹

Ultraviolet rays (UVs) are electromagnetic radiation with wavelengths ranging from 100 to 400 nm primarily derived from solar radiation and artificial light sources. Long-term or excessive exposure to UV (UVA and UVB) can directly damage the DNA of skin cells and is the main environmental risk factor of certain skin diseases such as sunburn, cutaneous lupus and skin cancer.⁹⁰ Since we cannot completely avoid the exposure of UVs in sunlight, it is inevitable that we will be exposed to both UV and BaP simultaneously in our daily lives (Table 4). An in vivo study indicated that long-term co-exposure to UVB and BaP effectively elevates skin cancer incidence in mice.⁹³ The joint skin toxicity of UV and BaP has been partially summarized in previous reviews focusing on studies published before 2009.^91,92 Mechanistically, BaP acts as a UVA photosensitizer to induce excessive ROS accumulation and subsequent oxidative DNA damage. Meanwhile, UVA facilitates intracellular BaP metabolism, promoting the formation of BPDE-DNA adducts.⁹² Such interactive effects trigger single- or double-strand DNA breaks and base substitution, thereby inducing H-ras gene mutations.⁹¹ Collectively, co-exposure synergistically enhances the mutation potential of key cancer-related genes (e.g., ras and p53), dysregulating downstream signaling pathways and protective enzymatic responses and ultimately promoting skin tumorigenesis. In addition to the consistent mechanisms (phototoxic, ROS production, oxidative stress, and DNA damage) described in earlier studies,^94,100 new evidence further indicated that impaired autophagy contributed to the damage and aging of human skin explants induced by UV (UVA + UVB) and BaP co-exposure.⁹⁹ BaP exhibits strong affinity for cutaneous mitochondria, triggering mitochondrial damage and lipid peroxidation.^98,100,101 Nevertheless, the skin toxicity induced by combined UV and BaP exposure remains controversial.⁹¹ Co-exposure to BaP and simulated sunlight (SSL) was found to suppress BaP-triggered upregulation of cutaneous cytochrome P450 genes and subsequent formation of DNA adduct, as UV radiation strongly inhibits BaP metabolic activation.^95,96,97 Such a discrepancy might be due to the difference in UV source, such as UV subtypes, wavelength spectra, irradiation intensity, and exposure duration, and the heterogeneity of experimental models across different species and cell types.

Table 4.

Toxicity caused by co-exposure of BaP and ultraviolet rays (UV)

Type	Experimental subjects (route of exposure)	Dosage and exposure duration	Lesion location/health impairment (+/−/n)	References
UVA	–	review	DNA lesions and accompanied mutations, skin cancer risk (+)	Toyooka et al.91; Burke et al.92
UVB	Swiss Albino mice	BaP (64 μg), UVB (180 mJ/cm2) 2 h later, thrice a week, 12 weeks	Skin cancer (+)	Das et al.93
UV (d-UV 300–400 nm) or UVA1 (350-400 nm)	normal human epidermal keratinocytes (NHEK)	BaP (1.5, 3, 6 nM)+ UV (7.5 J/cm2) for 3 consecutive days	Phototoxic (+), impaired keratinocyte clonogenic potential (+)	Soeur et al.94
UV (simulated sunlight, SSL)	human skin explants	BaP (0.1 μmol) + UV (minimal erythema dose (MED)/hour)	inhibition of BaP-induced expression of cutaneous cytochrome P450 genes and formation of DNA adducts (−)	von Koschembahr et al.95
UV (SSL)	skin explants from one human donor	SSL (6.4 J/cm2) + BaP (10 nmol, 24, 48, and 72 h)	UV strongly inhibited BaP metabolism (−)	Bourgart et al.96
UV (SSL)	human breast skin samples from healthy female donors	SSL (7.2 J/cm2), irradiated 1 h before or 24 h after the beginning of BaP (10 nmol) treatment.	inhibition of BaP-induced expression of cutaneous cytochrome P450 genes and formation of DNA adducts (−)	von Koschembahr et al.97
UVA	HaCaT and B16-F10 cell line	BaP (2 μΜ) for 48 h, then UVA (94, 282, 470, 658 and 940 mJ/cm2) irradiation	elevated oxidative stress (+); mitochondrial and DNA damage (+)	Gallego-Rentero et al.98
UVA (365 nm), UVB (312 nm)	three abdominal skin explants from patients	BaP (100 nM) + UVA (8 J/cm2) + UVB (10 mJ/cm2), treated on day 1, 2, 5, 6, collected on day 7.	downregulated autophagy (+)	Fayyad-Kazan et al.99
UVA	human diploid dermal fibroblasts of healthy women, HEK-293T cells	BaP (500 nM) + UVA (200 kJ/m2)	dysfunction of mitochondria (+) ROS accumulation and lipid peroxidation (+)	von Koschembahr et al.95; Larnac et al.100; Larnac et al.101

BaP, benzo[a]pyrene; HEK-293T, immortalized human embryonic kidney cells; HaCaT, human keratinocyte cell line; B16-F10, mouse melanocyte cell line; ROS, reactive oxygen species. +, synergistic or additive effect; -, antagonistic effect; n: no significant interactive effects.

Microwaves, as non-ionizing radiation, are widely applied in microwave heating, wireless communication, and radar systems. A long-term in vivo investigation exposed mice to 2450 MHz microwave irradiation combined with biweekly cutaneous BaP administration for six months. The results found that microwave radiation inhibited the cellular immune response in mice, resulting in the development of basal cell or squamous cell skin cancer.¹⁰²

Sulfur dioxide (SO₂)

Sulfur dioxide (SO₂) is a critical monitored indicator of ambient air pollution, which induces acute respiratory injuries after high-concentration or prolonged inhalation. In vivo animal experiments have verified the synergistic toxic effects of SO₂ and BaP co-exposure. Specifically, mice subjected to 5-day BaP treatment combined with 4-week SO₂ inhalation exhibited decreased mitochondrial membrane potential in hepatocytes, which further triggered cell apoptosis.¹⁰³ Another rodent study established a persistent inhalation co-exposure model, in which rats were continuously exposed to BaP (7 mg/m³)-adsorbed SO₂ (5 ppm) for 2 h per day across 9 consecutive days. On day 28 post co-exposure, it was found that the proliferation of cuboidal alveolar cells presented an initial increase followed by a subsequent decline.¹⁰⁴

The co-exposure research of BaP with electromagnetic radiation predominantly indicates their interaction effect in carcinogenesis, which is mainly concentrated on UV-related skin cancer with relatively well-elucidated molecular mechanisms. In comparison, relevant studies on the combined toxicity of BaP with other physical environmental pollutants, such as microwaves, X-rays, and radioactive particles, as well as air pollutant SO₂, remain limited and insufficient.

Outlook

Although the toxicological characteristics and carcinogenic risks of BaP have been extensively validated, it predominantly focuses on the toxicity of BaP itself. Notably, co-exposure scenarios of BaP with multiple environmental contaminants are frequently overlooked, which may lead to an inaccurate assessment of its real-world health hazards. The existing experimental studies of binary mixture exposure to BaP- pollutant cover pollutants including metals/metalloids, PMs, organic pollutants, and electromagnetic radiation. The toxic effects caused by co-exposure are mainly metabolic disorders, neurotoxicity, endocrine disorders, reproductive toxicity, genotoxicity, carcinogenicity, and liver and kidney damage. Nevertheless, the majority of relevant studies remain preliminary and lack systematic and in-depth exploration. However, prevailing evidence is derived from in vitro models, which fail to recapitulate the physiological complexity of human exposure scenarios. Given that environmental BaP contamination generally features low-dose and long-term persistent exposure, future toxicological assays should prioritize physiologically relevant experimental designs, including low-dose chronic and lifelong exposure models. Such improved models are essential to elucidate long-term latent toxic outcomes, developmental toxicity, and intergenerational toxic effects induced by BaP-based combined contamination.

Mechanistically, the combined toxicity of BaP and co-existing contaminants is primarily attributed to inflammatory activation, excessive ROS accumulation and subsequent oxidative stress, DNA damage, dysregulation of key signaling pathways (especially the CYP1A1-mediated metabolic pathway), and aberrant cellular apoptosis. However, the underlying molecular mechanisms remain insufficiently clarified. Current studies rely heavily on conventional single-target detection methods, while multi-omics profiling, computational toxicology, and systematic bioinformatics approaches are rarely integrated to decipher the mechanisms. Furthermore, epigenetic regulatory mechanisms governing BaP-combined toxicity remain largely uncharacterized, which hinders the comprehensive understanding of the upstream molecular signaling. To address these knowledge gaps, subsequent studies are required to establish systematic multi-dimensional research frameworks covering physiological simulation, molecular mechanism exploration, and multi-technology cross-validation. The understanding of interactive toxic effects and underlying mechanisms of BaP co-exposure will complement the theoretical basis of BaP environmental toxicology and provide robust scientific support for accurate human health risk assessment and contamination risk management.

Acknowledgments

The present study was supported by the Natural Science Foundation of Jiangsu Province (BK20220664) and Xuzhou Medical University start-up grant for excellent scientists (D2020020).

Author contributions

W.L. and X.Y. carried out the concept and design of the review. H.Y., X.Z., and Y.S. participated in the acquisition and interpretation of data. H.Y. drafted the manuscript. W.L. and X.Y. supervised this work. All authors read and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.