Polychlorinated Biphenyls

From 2579 boys, aged 10–16 years in 1999, enrolled in an earlier pilot study to generate growth and maturation curves for boys in Chapaevsk [13 ], a subset of 246 older boys (14.0 to 16.9 years) were identified for a sub-study in which blood samples and questionnaire information were obtained. Older boys were chosen for study because blood samples were required and participation rates were expected to be higher than among younger children. Of the 246 boys, 221 had blood samples collected, and of these samples, 30 bloods were initially sent to the CDC for chemical analysis of dioxins, furans and PCBs. By design, of the 30 blood samples, 15 were from children with cryptorchidism or hypospadias, and 15 were from children with neither condition (controls). The selection of the 15 cases and 15 controls was done blindly in relation to factors that may predict dioxin levels.
Each of the 30 boys, with his mother, was asked to complete a nurse-administered detailed questionnaire on medical history, diet, and lifestyle. The diet questions were used to measure the current and lifetime consumption of locally grown or raised foods. The question was worded, "Does your child eat any of the following foods from local sources (i.e. your own garden or farms or lakes in the Chapaevsk area)? Yes/No". There were separate questions for current intake and lifetime intake of each food item. The distances the boys lived from the Khimprom factory at the time of the study and during pregnancy were assessed by questionnaire based on maternal self-report as <2, 2–6, or >6 kilometers, and the distance at the time of the study was also estimated using ArcView GIS 3.0 mapping of addresses.

The CCLS is a population-based case-control study in 35 counties in California, including 17 counties in the San Francisco Bay area and 18 in the Central Valley [11 (link),26 (link)]. Between 1995 and 2012, cases ≤14 years old were ascertained within 72 h of diagnosis from nine major pediatric clinical centers in the study area. Using California birth certificate information, controls were matched to cases on the basis of date of birth, sex, Hispanic ethnicity, and maternal race. Parents of both case and control participants were initially interviewed to gather information about their child’s exposure to suspected leukemia risk factors. Families who had not moved since the child’s diagnosis date (reference date for controls) were eligible for a second interview (Tier 2), during which carpet dust samples were collected and information about pesticide use was obtained. The second interview and dust sampling were limited to cases and controls <8 years old (diagnosed December 1999 to June 2006) to ensure the samples reflected early-life chemical exposure of the child. Case-control matching was not maintained due to residential eligibility criteria and voluntary participation. There were 731 participants for the Tier 2 interviews (324 cases and 407 controls). Of these, 296 cases (91%) and 333 controls (82%) agreed to participate. Due to insufficient dust or interferences in the chemical analyses, some samples were not quantified, leading to a final 277 cases and 306 controls (n = 583) [13 (link)].
The dust samples were collected using either a high-volume small surface sampler (HVS3) or a household vacuum cleaner. As described previously [26 (link)], concentrations of 64 organic chemicals (ng/g dust) were measured using gas chromatography/mass spectrometry (GC/MS) in multiple ion-monitoring mode after extraction with three different extraction methods. Nine metals were measured using microwave-assisted acid digestion combined with inductively coupled plasma/mass spectrometry (ICP/MS).
Our analysis investigated the association of 67 chemicals with the risk of childhood leukemia. Out of the entire CCLS dataset, only chemical exposure variables with at least 20% non-missing observations were included, as past experience has shown that higher levels of missingness contribute negligible information on potential relations with an outcome. We organized exposures into seven chemical class indices: polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), insecticides, herbicides, metals, tobacco exposure markers of nicotine and cotinine, and polybrominated diphenyl ethers (PBDEs). The reason for these groupings was that the chemicals share a structural similarity (e.g., PCBs, PAHs, metals) or usage (e.g., herbicides, insecticides). Most missing data were the result of findings below a chemical’s detection limit. Additionally, there were missing data in the PBDE chemicals due to insufficient levels of house dust for analysis. Both types of missing observations were imputed with log-normal distributions, as previously described [15 (link)].
The interviewers took a global positioning system reading of the residence coordinates, which were linked to census data for the computation of the neighborhood deprivation index and used to evaluate spatial random effects for unexplained spatial heterogeneity in leukemia risk.

The surface MEA chip and flexible MEAs were fabricated following previous procedures with small modifications [34 (link)]. A Si wafer with a 1µm thick SiO₂ layer (University Wafer Inc., South Boston, MA, USA) was first cleaned by sonicating in acetone, isopropanol, and DI water sequentially for 5 min each step. The wafer was then dried on a hot plate at 150 °C for 3 min, then the surface was cleaned and activated by O₂ plasma using a reactive ion etcher (RIE, Trion Phantom III LT, Clearwater, FL, USA) for 120 s at 200 mTorr pressure and 150 Watts power. The wafer was then spin-coated with SU-8 2015 (MicroChemicals, Ulm, Germany) at 5000 rpm for 1 min and soft baked at 65 °C for 3 min and 95 °C for 5 min to evaporate solvent, then the wafer was exposed using a maskless aligner (MLA, MLA100, Heidelberg Instruments, Heidelberg, Germany) with a dose of 400 mJ/cm². After exposure, the SU-8 first layer was post-baked at 65 °C for 3 min and 95 °C for 5 min, developed using SU-8 developer (MicroChemicals, Germany) for 1 min, and cleaned by isopropanol and DI water, Then, the wafer was hard baked at 200 °C, 180 °C, and 150 °C for 5 min each for SU-8 curing and allowed to cool down below 95 °C (step skipped for in vitro MEA fabrication). The wafer was then treated with O2 plasma to clean, activate, and roughen the SU-8 with RIE for 75 s at a pressure of 200 mTorr and 150 W power. The treated wafer was then spin-coated with AZ P4620 photoresist (MicroChemicals, Germany) at 5000 rpm for 1 min and baked at 105 °C for 5 min for resist curing. After baking, the wafer was exposed using MLA with a dose of 700 mJ/cm², then developed using AZ400k 1:4 developer (MicroChemicals, Germany), cleaned by water rinse, and dried with N2 gas flow. A mild 120 s RIE O2 plasma treatment at pressure 600 mTorr and 60 W power was performed to clean the SU-8 before metal deposition. A 10 nm Ti adhesion layer and 100 nm Au layer were evaporated on the wafer using an Electron Beam Evaporator Plassys MEB550S (Marolles-en-Hurepoix, France). The metal was then lifted off in acetone overnight. The next day, the wafer was first rinsed with water, dried under N₂ flow, and cleaned by O2 plasma for 60 s at 600 mTorr and 60 W, then spin-coated with SU-8 2015 for insulation layer at 5000 rpm for 1 min and soft baked at 65 °C for 2 min and 95 °C for 5 min. The wafer was then exposed using MLA with a dose of 400 mJ/cm², post-backed, and developed with an SU-8 developer. The wafer was then cleaned with isopropanol and water, and was hard baked at 200 °C, 180 °C, and 150 °C for 5 min each to fully cure SU-8 and allowed to cool down below 95 °C. For in vitro use, the wafer was then cut into rectangular pieces (1.5 × 2 cm) containing an MEA and used for in vitro experiments. For in vivo use, the flexible MEAs were released from the wafer using buffered oxide etchant (1:7) (MicroChemicals, Germany) in an acid hood for 8 h to etch away the SiO₂ layer. Customized PCBs were mounted with ZIF and Omnetics connectors. ZIF connectors were used to interface with flexible MEAs, and Omnetics connectors were used to interface with the potentiostat and electrophysiology recording system.